Parallelized sample processing and library prep

ABSTRACT

Described herein are methods, kits and systems for sample enrichment, multi-step library preparation, sample normalization, detection of sample biomolecules and combinations thereof. Enrichment and multi-step library preparation is described in the context of microfluidic workflows. Sample barcoding methods and kits are described for increasing sample throughput while reducing background in negative samples. Integrated microfluidic devices comprising sample processing unit cells coupled to an array of reaction sites are provided for integrated workflows.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 63/049,998, filed Jul. 9, 2020; U.S. ProvisionalApplication No. 62/979,832, filed Feb. 21, 2020; and U.S. ProvisionalApplication No. 62/979,209, filed Feb. 20, 2020, the entire contents ofall of which are incorporated herein by reference for all purposes.

BACKGROUND

Automated microfluidic systems and/or parallel library preparation ofsamples can reduce labor, reagent use, and variability in sampleprocessing. These benefits are further improved by sample indexing(barcoding), which allows samples processed in parallel to be combinedprior to sequencing or detection by qPCR. In additional, a more fullyintegrated microfluidic workflow would reduce hands-on time and humanerror.

SUMMARY

As described herein, an integrated microfluidic device may thereforeinclude, an array of reaction sites and a plurality of sample processingunit cells including a plurality of sample processing sites, wherein theunit cell is in fluidic communication with a plurality of differentreagent inlets, and wherein sample inlets to the array are downstream ofthe plurality of sample processing sites of the plurality of unit cells.

The plurality of reagent inlets may share a common channel to each unitcell. The microfluidic device may include a multiplexor configured tocontrol which reagent inlet is used to load a processing site of theunit cell.

The plurality of sample processing sites may include a plurality ofloops and/or chambers. Each unit cell further includes one or more of asample inlet channel, a waste outlet channel, additional reagent inlets,and/or additional columns.

Each unit cells may include a plurality of valves configured to controlthe unit cell. The plurality of valves may be configured to deliversample and reagents to different locations in the unit cell. Theplurality of valves may be configured to place sample processinglocations in isolation or in communication with one another. Theplurality of valves may be configured to drive mixing at differentlocations. The plurality of valves are configured to direct flow ofsample or reagents solution out of the unit cell For example, the unitcell includes a peristaltic pump (e.g., defined by a set of valves inseries).

Wherein individual unit cells further includes at least one columnconfigured to retain beads. The column may include a sieve architectureproviding a plurality of openings through which fluid may flow but beadslarger than the outlet opening may be retained.

In certain aspects, an integrated microfluidic device may include: anarray of reaction sites; and a plurality of sample processing unit cellsincluding a plurality of sample processing sites, wherein the unit cellis in fluidic communication with a plurality of different reagentinlets; wherein sample inlets to the array are downstream of theplurality of sample processing sites of the plurality of unit cells.

A method may include loading beads into a column of a unit cell andcapturing sample (i.e., biomolecules of a sample such as proteins,antibodies, RNA, viral particles, etc.) on the bead (e.g., before orafter loading the beads into the column). As discussed herein, the beadmay include (e.g., present on its surface) one or more of a protein(e.g., an antibody, such as an antibody to a target serum protein orviral antigen) and oligonucleotide (e.g., that hybridizes to target RNA,such as a viral RNA). Additional steps may include washing beads, suchthat a wash buffer flows over the beads in the column and into a wasteoutlet. Optionally a reporter, such as an oligonucleotide-conjugatedantibody that binds to target biomolecules or a oligonucleotide probethat hybridizes to target biomolecules may be flowed over the beads.Additional steps may include eluting from the beads, such as by flowingan elution buffer over the beads in the column and optionally furthercycling the elution buffer across the beads such as by passing thebuffer around a loop using a peristaltic pump.

Sample barcoding for multiplexing may increase sample throughput but theleftover primers (e.g., from sample with little or no target) may createcrosstalk, leading to a false positive and/or higher background.Discussed herein are methods and kits for reducing such crosstalk.

In certain aspects, an assay method for detecting at least one targetnucleic acid in a plurality of samples includes:

-   -   a) reverse transcribing and preamplifying a target nucleotide        sequence in each of S separate samples to produce a tagged        target nucleotide sequence from each sample,        -   wherein at least one of the S samples includes the target            nucleotide sequence,        -   wherein the tagged target nucleotide sequence includes a            sample tag and a target nucleotide sequence,        -   wherein preamplifying is with a tagged target-specific            primer that includes a sample tag and a target-specific            sequence, and        -   wherein the target-specific sequence hybridizes to a portion            of the target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) splitting the mixture into a plurality of reaction sites;    -   d) adding different primer pairs to each reaction sites;    -   e) amplifying the tagged target nucleotide sequence from a        different sample in each reaction site,        -   wherein each different primer pair includes a primer that            hybridizes to a different sample tag; and/or    -   f) detecting the presence of the of the amplified tagged target        nucleic acid by qPCR with a fluorescent target-specific probe        that includes at least a portion of the target-specific sequence        but does not include a sample tag;        -   wherein step e of amplifying is in the presence of the            target-specific probe.

More generally, an assay method for detecting at least one targetnucleic acid in a plurality of samples may include:

-   -   a) separately subjecting each of S samples to an encoding        reaction that produces a tagged target nucleotide sequence using        at least one tagged target-specific primer, wherein at least one        of the S samples includes the target nucleotide sequence and        wherein the tagged target nucleotide sequence includes a sample        tag and a target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) splitting the mixture into a plurality of reaction sites;    -   d) adding different primer pairs to different reaction sites,        wherein each different primer pair includes a primer that        hybridizes to a different sample tag to amplify a tagged target        nucleotide sequence from a specific sample;    -   e) amplifying the tagged target nucleotide sequence from the at        least one of the S samples in the presence of a target-specific        probe, wherein the target-specific probe includes a sequence        identical to at least a portion of a target-specific sequence of        the target-specific primer but does not include a sample tag;        and/or    -   f) detecting the presence of the tagged target nucleotide.

An assay method for detecting at least one target nucleic acid in aplurality of samples may include:

-   -   a) separately subjecting each of S samples to an encoding        reaction that produces a tagged target nucleotide sequence using        at least one tagged target-specific primer, wherein at least one        of the S samples includes the target nucleotide sequence and        wherein the tagged target nucleotide sequence includes a sample        tag and a target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) amplifying the tagged target nucleotide sequence from the at        least one of the S samples in the presence of a target-specific        probe, wherein the target-specific probe includes a sequence        identical to at least a portion of a target-specific sequence of        the target-specific primer but does not include a sample tag;        and/or    -   d) detecting the presence of the tagged target nucleotide.        -   Also described herein are kits for performing any of the            methods described herein.

Aspects of the subject application also include methods, kits anddevices for parallel processing of samples, such as for librarypreparation and/or normalization.

In some embodiments, a method of library normalization includes one ormore of:

-   -   a. obtaining aliquots from a plurality of samples, wherein        samples polynucleotides have spaced inverted repeats;    -   b. performing suppression PCR on the aliquots of step a;    -   c. quantifying amplification products from step b;    -   d. pooling the plurality of samples to form a library normalized        based on the quantification of step c; and/or        -   wherein the pooled plurality of samples have not undergone            the suppression PCR of step b.

Methods of the above embodiments may further include aspects of sampletype, sample number, suppression PCR, polynucleotide characteristics,sample enrichment and/or preparation, primers, sample quantitationand/or normalization, microfluidic devices, metrics of improvement,and/or sequencing applications as described herein.

In some embodiments, a kit for library quantification of polynucleotidesby suppression qPCR may include a primer including a sequence identicalto at least 8 nucleotides of one of the inverted repeats of anotherpolynucleotide in the kit, such as a library quantification standardhaving spaced inverted repeats separated by at least 150 nucleotides.

In some embodiments, a kit for library preparation and quantificationmay include adaptors (or primers) together providing an inverted repeatat least 8 nucleotides in length (e.g., capable of producingpolynucleotides with inverted repeats flanking inserts); and/or a primerincluding a sequence identical to at least 8 nucleotides of an invertedrepeat.

Kits of any of the above embodiments may further include, or providesupport for performing methods that include, a sample type, samplenumber, reagents for suppression PCR, polynucleotide characteristics,reagent for sample enrichment and/or preparation, primers, reagents forsample quantitation and/or normalization, microfluidic devices, metricsof improvement, and/or sequencing applications as described herein. Kitsmay include beads, microfluidic devices, reverse transcription reagents,primers and/or master mix for PCR (such as suppression PCR) reagents,dye (such as a passive reference dye).

In general, aspects of the subject application may include one or moreof: a kit for performing any of the method aspects above, a method oflibrary normalization based on suppression qPCR, a method of suppressionqPCR, a method of sequencing a library normalized by suppression qPCR,and/or a pool of samples normalized based on suppression qPCR of any ofthe method aspects above.

In certain aspects, a method of parallel sample processing includessplint ligation in which a target nucleic acid is the splint template.

For example, a method of processing a splint hybridization product mayinclude hybridizing a first probe and a second probe to a target nucleicacid to form a hybridization product, where a 3′-OH end of the firstprobe adjacent to a 5′-PO4 end of the second probe. At least one of thefirst probe and the second probe comprises a binding moiety. The methodmay further include capturing the hybridization product by specificallybinding the binding moiety to a solid support.

In another example, a method of detecting a splint ligation product mayinclude, hybridizing a first probe and a second probe to a targetnucleic acid to form a hybridization product, wherein a 3′-OH end of thefirst probe is adjacent to a 5′-PO4 end of the second probe. The methodmay further include ligating the first probe and second probes to form aligation product. The method may further include detecting the presenceof the ligation product.

Hybridization product or ligation product may be captured on a solidsupport (e.g., beads such as beads in a column of a microfluidic device)as described further herein. Splint ligation probes may comprise one ormore sample barcode sequences, allowing for pooling of samples,processing of samples together (e.g., capture, ligation and/orpreamplification) prior to splitting the pool and separately detectingligation products of different samples (e.g., on an array IFC). Thetarget nucleic acid may be DNA or an RNA, such as a viral genomic RNA ora mammalian gene transcript.

In certain aspects, a kit for parallel sample process may includereagent for splint ligation methods described herein. Such a kit mayhave two splint ligation probes described in any embodiments herein, andmay optionally further comprise ligase for forming ligation products,primers for amplifying ligation products, and/or additional reagentssuch as reagents for separating ligation product from solid support.Splinted ligation kits may further include one or more microfluidicdevices described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an abstraction of a microfluidic device 100 having at leastsample inlet(s) 104 and reagent inlet(s) 106 that feed into a fluidiccircuit 102. Such a device may perform sample preparation stepsdescribed herein. The device may include additional inlets and outlets.

FIG. 2 is an image of an exemplary elastomeric microfluidic device 200of the subject application. FIG. 2A shows the device itself, an 48.AtlasIFC (Fluidigm). FIG. 2B shows the device overlaid with markingsindicating sample inlets 204, sample barcode reagent inlets 206, commonreagent inlets 208, wash solution inlets 210, as well as waste outlets212. Inlets can be loaded onto the microfluidic through backpressureapplied by a pneumatic system (controller). Control ports 214 are alsomarked, and can be pressurized by the pneumatic system to operateelastomeric valves on the device to direct flow and fluidiccommunication. A substrate underlying the microfluidic circuit thermallycouples to a thermocycler.

FIG. 3 is an abstraction of the architecture of a unit cell 320, e.g.,of a fluidic circuit of FIG. 1 or 2. The unit cell may process a singlesample provided by a sample inlet 304, and provide multiple sampleprocessing sites 314 in which the sample is reacted with differentreagents provided by one or more reagent inlet(s) 306. The unit cell mayinclude a column 308for enriching sample in the unit cell and/oradditional elements such as waste outlets, valves, pumps, or othercomponents of microfluidic devices described herein.

FIG. 4 is a detailed schematic of an exemplary unit cell 402 of FIG. 2.A plurality of reagent inlet channels 406 are shown, which may share achannel joining the unit cell. The plurality of sample processing sitesmay have chambers 414 and/or sample processing loops 412 for mixingsample and/or reagents between chambers. A waste outlet channel 416 isshown for removing excess or unwanted fluid from the unit cell. Valves(not shown) may be configured along the unit cell and operated todeliver sample and reagents to different locations (e.g., column,chambers, loops) in the unit cell, place sample processing loops orchambers in isolation or in communication with one another, drive mixingbetween reagents and/or sample at different locations, and direct flowof sample or reagent solution out of the unit cell (e.g., to wasteand/or to a harvest outlet). The unit cell may include a sample inletchannel 404, a column 408 for enriching sample in the unit cell, and/oradditional elements such as valves, pumps, or other components ofmicrofluidic devices described herein.

FIG. 5 shows the principal mechanism of suppression PCR in which a shortsequence with spaced inverted repeats, such as ITRs, forms a hairpinthat suppresses amplification by a primer that hybridizes to theinverted repeats exposed by the longer DNA molecule.

FIG. 6 shows various polynucleotides produced in certain librarypreparation workflows, such as through PCR based incorporation ofinverted repeats, sequencing adapters and/or sample barcodes. A desiredlibrary product (top) with spaced inverted repeats (circled) that flankan insert which may comprise more than half the length of thepolynucleotide. The polynucleotide further includes a sequencing adaptor(dotted line). A primer dimer product (middle) is shown that hasinverted repeats in close proximity. Such a product may interfere withtraditional qPCR. A “bubble DNA” product (bottom) is shown in whichadapters have reannealed and the inserts are mismatched. Such a productmay interfere with quantification of long products (e.g., by a mobilityassay such as capillary electrophoresis).

FIG. 7 shows a microfluidic multi-step sample preparation workflow (top)that provides sample barcoded libraries. A qPCR quantification andnormalization workflow to guide pooling of the different samplesdifferent samples is shown (bottom). In the subject application, theqPCR step may be a suppression qPCR as described herein.

FIG. 8 shows read uniformity for unnormalized vs normalized samplepools. The normalized sample pools were pooled based on quantificationby suppression qPCR. Traditional normalization (e.g., by bioanalyzer orby traditional qPCR) tends to show similarity to the unnormalizedsample.

FIG. 9 shows number of genes detected for unnormalized vs normalizedsample pools. The normalized sample pools were pooled based onquantification by suppression qPCR. Traditional normalization (e.g., bybioanalyzer or by traditional qPCR) tends to show similarity to theunnormalized samples.

FIGS. 10A and 10B shows an exemplary splint hybridization product andsplint ligation product respectively.

FIG. 11 shows exemplary splinted ligation workflows.

FIG. 12 is a schematic of an array integrated fluidic circuit (IFC).

FIG. 13 is an image of an exemplary elastomeric microfluidic device andexemplary loading scheme of the subject application.

FIG. 14 is schematic similar to that of FIG. 3 and showing directions offlow from inlets, outlets and within the unit cell such as in a loadingscheme of FIG. 13.

FIG. 15 is a schematic showing exemplary loading schemes for RNAsequencing preparation (A) and DNA sequencing preparation (B).

FIG. 16 is a schematic showing exemplary loading schemes foroligonucleotide detection on chip (such as detection of a viral RNA) (A)and sample preparation for detection of a protein (such as a cancermarker, viral antigen, or antibody to a viral antigen) (B).

FIG. 17 is a schematic similar to that of FIG. 3 and showing anexemplary unit cell with a plurality of columns.

FIG. 18 shows an exemplary cleanup step.

FIG. 19 shows an exemplary capture, sample preparation and PCRamplification.

FIG. 20 shows a multiplex sample barcoding workflow of the subjectapplication.

FIG. 21 shows a simple Dorfman pooling method.

FIG. 22 shows the efficiency of the multiplex sample barcoding (mpe) andDorfman pooling (pe) methods when 4 samples are mixed (A) or 8 samplesare mixed (B).

FIG. 23 shows crosstalk that can occur when using the multiplexed samplebarcoding approach of FIG. 20.

FIG. 24 provides a reaction scheme in which leftover primer from anegative sample (i.e., sample B that does not have a target nucleotidesequence) may react with the preamplified target nucleotide sequencefrom a positive sample (sample A).

FIG. 25 provides a reaction scheme in which target specific probecompetes with leftover primer.

FIG. 26 shows qPCR curves under the scheme of FIG. 24.

FIG. 27 shows qPCR curves under the scheme of FIG. 25, demonstrating aCT increase of 2 for negative samples compared to FIG. 26.

FIG. 28 shows another approach to reducing cross talk.

DETAILED DESCRIPTION

Methods, microfluidic systems and kits for sample preparation, includinglibrary preparation and normalization, are provided herein. Someembodiments may provide for specific sequencing applications includingmRNA sequencing applications or DNA sequencing applications describedherein.

The methods, systems, and kits may include microfluidic devices and/orcontrollers for enrichment and multi-step sample preparation.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. For convenience, certain termsare highlighted, for example using italics and/or quotation marks. Theuse of highlighting has no influence on the scope and meaning of a term;the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification, including examples of any terms discussed herein, isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to the preferred embodiments.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range. Numerical quantities given herein areapproximate, meaning that the term “about” or “approximately” can beinferred if not expressly stated.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes for examplepolypeptides and polynucleotides.

The term “polymer” means any substance or compound that is composed oftwo or more building blocks (‘mers’) that are repetitively linked toeach other. For example, a “dimer” is a compound in which two buildingblocks have been joined together.

The term “polynucleotide” (also referred to as oligonucleotides) as usedherein refers to a polymeric molecule having a backbone that supportsbases capable of hydrogen bonding to typical polynucleotides, where thepolymer backbone presents the bases in a manner to permit such hydrogenbonding in a sequence specific fashion between the polymeric moleculeand a typical polynucleotide (e.g., single-stranded DNA). Such bases aretypically inosine, adenosine, guanosine, cytosine, uracil and thymidine.Polymeric molecules include double and single stranded RNA and DNA, andbackbone modifications thereof, for example, methylphosphonate linkages.In the context of a sample for library normalization, a polynucleotidemay refer to a sample indexed (or barcoded) polynucleotide. Such apolynucleotide may also have sequencing adaptors flanking an “insert”sequence derived from mRNA or gDNA.

Thus, a “polynucleotide” or “nucleotide sequence” is a series ofnucleotide bases (also called “nucleotides”) generally in DNA and RNA,and means any chain of two or more nucleotides. A nucleotide sequencetypically carries genetic information, including the information used bycellular machinery to make proteins and enzymes. These terms includedouble or single stranded genomic and cDNA, RNA, any synthetic andgenetically manipulated polynucleotide, and both sense and anti-sensepolynucleotide (although only sense stands are being representedherein). This includes single- and double-stranded molecules, i.e.,DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids”(PNA) formed by conjugating bases to an amino acid backbone. This alsoincludes nucleic acids containing modified bases, for examplethio-uracil, thio-guanine and fluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.). Polynucleotides may contain one or more additional covalentlylinked moieties, such as, for example, proteins (e.g., nucleases,toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators(e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactivemetals, iron, oxidative metals, etc.), and alkylators. Thepolynucleotides may be derivatized by formation of a methyl or ethylphosphotriester or an alkyl phosphoramidate linkage. Furthermore, thepolynucleotides herein may also be modified with a label capable ofproviding a detectable signal, either directly or indirectly. Exemplarylabels include radioisotopes, fluorescent molecules, biotin, and thelike.

“DNA” (deoxyribonucleic acid) means any chain or sequence of thechemical building blocks adenine (A), guanine (G), cytosine (C) andthymine (T), called nucleotide bases, that are linked together on adeoxyribose sugar backbone. DNA can have one strand of nucleotide bases,or two complimentary strands which may form a double helix structure.“RNA” (ribonucleic acid) means any chain or sequence of the chemicalbuilding blocks adenine (A), guanine (G), cytosine (C) and uracil (U),called nucleotide bases, that are linked together on a ribose sugarbackbone. RNA typically has one strand of nucleotide bases.

A “polypeptide” or “protein” (one or more peptides) is a chain ofchemical building blocks called amino acids that are linked together bychemical bonds called peptide bonds. A protein or polypeptide, includingan enzyme, may be “native” or “wild-type”, meaning that it occurs innature; or it may be a “mutant”, “variant” or “modified”, meaning thatit has been made, altered, derived, or is in some way different orchanged from a native protein, or from another mutant.

A “probe” in the context of an oligonucleotide reaction (e.g., encodingreaction, reverse transcription, amplification, and so forth) referssimply to an oligonucleotide sequence that binds to (hybridizes) atarget. Described herein are splint ligation probes and competitionprobes do not necessarily provide a signal when bound to a targetnucleotide sequence. However, qPCR probes, or probes described as havinga fluorophore (or fluorophore and quencher), may be used to detect atarget.

A “sample biomolecule of interest” is a target, often a specificoligonucleotide or protein, that may be specifically bound, processedand/or detected in an assay as described herein.

The term “flow” means any movement of liquid or solid through a deviceor in a method of the invention, and encompasses without limitation anyfluid stream, and any material moving with, within or against thestream, whether or not the material is carried by the stream. Forexample, the movement of molecules or cells through a device or in amethod of the invention, e.g. through channels of a microfluidic chip ofthe invention, comprises a flow. This is so, according to the invention,whether or not the molecules or cells are carried by a stream of fluidalso comprising a flow, or whether the molecules or cells are caused tomove by some other direct or indirect force or motivation, and whetheror not the nature of any motivating force is known or understood. Theapplication of any force may be used to provide a flow, includingwithout limitation, pressure, capillary action, electroosmosis,electrophoresis, dielectrophoresis, optical tweezers, and combinationsthereof, without regard for any particular theory or mechanism ofaction, so long as molecules or cells are directed for detection,measurement or sorting according to the invention.

An “inlet region” is an area of a microfabricated chip that receivesmolecules or cells for detection measurement or sorting. The inletregion may contain an inlet channel, a well or reservoir, an opening,and other features which facilitate the entry of molecules or cells intothe device. A chip may contain more than one inlet region if desired.The inlet region is in fluid communication with the main channel and isupstream therefrom.

An “outlet region” is an area of a microfabricated chip that collects ordispenses molecules or cells after detection, measurement or sorting. Anoutlet region is downstream from a discrimination region, and maycontain branch channels or outlet channels. A chip may contain more thanone outlet region if desired.

A “loop” or “sample processing loop” is a looped channel that may beoperated (e.g., by dilation pumping or peristaltic pumping) to mixsolution in the loop. Loops may be dynamic, such that valves areoperated to define loops and put different chambers in communicationwith one another. The loop may have any shape. The channel or channelscomprising a loop may have or cooperate with pumps and/or valves to openand close the loop, and/or to provide or drain contents to and from theloop. In certain embodiments, the loop can be isolated or closed fromother channels in a microfluidic device. Also in certain embodiments,fluid can be circulated in the loop, for example by providing aperistaltic pump comprising three or more microvalves.

In certain embodiments, a “circulation loop” is located within the chip,typically in or communicating with a unit cell, in which a fluid (e.g.the flow of a biological sample) is circulated. The circulation loop maycomprise a “hybridization loop” or “target loop” in which the flow isdirected past a series of targets or probes (e.g. DNA or proteins) thatare in or exposed to the loop and its contents, such as in a column. Forexample, probes may be patterned on the surface of a substrate or beads,e.g. a solid substrate and also called a “probe substrate”.

A “detection region” is a location within the chip, typically in orcoincident with the main channel (or a portion thereof) and/or in orcoincident with a detection loop, where molecules or cells to beidentified, characterized, hybridized, measured, analysed or sorted(etc.), are examined on the basis of a predetermined characteristic. Ina preferred embodiment, molecules or cells are examined one at a time.In other preferred embodiments, molecules, cells or samples are examinedtogether, for example in groups, in arrays, in rapid, simultaneous orcontemporaneous serial or parallel arrangements, or by affinitychromatography. In one such embodiment, a sample is exposed to probes indetection region, preferably probes having a predetermined patternwithin or coincident with a detection region, e.g. a targethybridization or detection loop. Preferably, the molecule or cellcharacteristic is detected or measured optically, for example, bytesting for the presence or amount of a reporter. For example, thedetection region is in communication with one or more microscopes,diodes, light stimulating devices, (e.g., lasers), photomultipliertubes, and processors (e.g., computers and software), and combinationsthereof, which cooperate to detect a signal representative of acharacteristic, marker, or reporter, and to determine and direct themeasurement or the sorting action at the discrimination region. Insorting embodiments, the detection region is in fluid communication witha discrimination region and is at, proximate to, or upstream of thediscrimination region.

A “discrimination region” or “branch point” is a junction of a channelwhere the flow of molecules or cells can change direction to enter oneor more other channels, e.g., a branch channel, depending on a signalreceived in connection with an examination in the detection region.Typically, a discrimination region is monitored and/or under the controlof a detection region, and therefore a discrimination region may“correspond” to such detection region. The discrimination region is incommunication with and is influenced by one or more sorting techniquesor flow control systems, e.g., electric, electro-osmotic, (micro-)valve, etc. A flow control system can employ a variety of sortingtechniques to change or direct the flow of molecules or cells into apredetermined branch channel.

A “branch channel” is a channel which is in communication with adiscrimination region and a main channel. Typically, a branch channelreceives molecules or cells depending on the molecule or cellcharacteristic of interest as detected by the detection region andsorted at the discrimination region. A branch channel may be incommunication with other channels to permit additional sorting.Alternatively, a branch channel may also have an outlet region and/orterminate with a well or reservoir to allow collection or disposal ofthe molecules or cells.

A “gene” is a sequence of nucleotides which code for a functionalpolypeptide. For the purposes of the invention a gene includes an mRNAsequence which may be found in the cell. For example, measuring geneexpression levels according to the invention may correspond to measuringmRNA levels. “Genomic sequences” are the total set of genes in anorganism. The term “genome” denotes the coding sequences of the totalgenome.

Polynucleotides may “hybridize” to each other when at least one strandof one polynucleotide can anneal to another polynucleotide under desiredor defined stringency conditions. Stringency of hybridization isdetermined, e.g., by a) the temperature at which hybridization and/orwashing is performed, and b) the ionic strength and polarity (e.g.,formamide) of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two polynucleotides containsubstantially complementary sequences; depending on the stringency ofhybridization, however, mismatches may be tolerated. Typically,hybridization of two sequences at high stringency (such as, for example,in an aqueous solution of 0.5×SSC at 65° C.) requires that the sequencesexhibit some high degree of complementarity over their entire sequence.Conditions of intermediate stringency (such as, for example, an aqueoussolution of 2×SSC at 65° C.) and low stringency (such as, for example,an aqueous solution of 2×SSC at 55° C.), require correspondingly lessoverall complementarity between the hybridizing sequences. (1×SSC is0.15 M NaCl, 0.015 M Na citrate.) Polynucleotide sequences that“hybridize” to the polynucleotides herein may be of any length. In oneembodiment, such polynucleotide sequences are at least 10, at least 15,or at least 20 nucleotides long.

A sample “barcode”, “tag” or “index” as used herein may beinterchangeable. In the context of an encoding reaction, such as withsample tagged (i.e., barcoded) primers, a tag (i.e., barcode) is asequence identifying a sample, such that the sample may thereafter bepooled with other samples while still identifying reaction products thatcame from each sample. As such, a sample tagged primer comprises asequence identifying the sample it amplifies a target nucleotidesequence from. For example, sequencing can read the sample tags (i.e.,indexes) to identify which sample a target read came from. Aspects ofthe application include selective amplification of sample taggednucleotide sequences, such as by qPCR using at least one primer to asample tag (that hybridizes a sample tag sequence).

An “identical sequence” means sequences, usually 6 nucleotides orlonger, which is identical to one another. When one sequence is of aoligonucleotide such as a target nucleotide sequence that has beenamplified, either strand may be considered when determining if thesequence is identical (e.g., to a primer or probe).

Sample

In certain aspects, at least 8, 12, 24, 48, 96, or 384 samples areprocessed by the subject methods or kits. Samples may be from anybiological source, such as eukaryotic sample (e.g., human, primate,rodent) or a bacterial sample. Samples may have biomolecules ofinterest, such as nucleic acids (e.g., polynucleotides) describedherein. Samples may be derived from a cell sample, such as a tissuesample or cell culture. In certain aspects, samples may have beenderived from a fixed tissue (e.g., solid tissue or cells). In certainaspects, fixed tissue (such as FFPE tissue) may have undergonefragmentation (e.g., of RNA) and be of variable quality that wouldresult in variable sequencing depth. In certain aspects, such as when atarget biomolecule is a biomarker or a viral antigen, the sample maycomprise a blood sample (e.g., serum, plasma or whole blood), a salivasample, or a nasal swab. Nucleic acids may be present in the sample inthe absence or a minimal amount of protein. Aspects include providing orproducing sample libraries in which sample polynucleotides are at leastpartially prepared for sequencing, such as by addition of sequencingadaptors.

Sequencing Technologies

Some embodiments may provide for specific sequencing applicationsincluding mRNA sequencing applications (such targeted RNA-seq, 3′RNA-seq, full-length RNA sequencing) or DNA sequencing applications(such as whole genome sequencing (WGS), targeted resequencing, chromatinimmunoprecipitation (ChIP) sequencing, RNA immunoprecipitation(RIP-Seq), chromatin accessibility sequencing (ATAC-seq)) andepigenetics such as methylation sequencing (bisulfite sequencing). Anysuitable sequencing technique discussed herein or known in the art iswithin the scope of the subject application.

Certain sequencing methods, and corresponding library preparationworkflows, are known in the art, including workflows that allow forsample barcoding (e.g., through dual indexing). For example, an IlluminaAdapter Sequences listing published by Illumina provides adaptersequences, index sequences, and primers, for common library prep kitsincluding Nextera kits, AmpliSeq, TruSight, and TruSeq kits. These andother sequencing methods and library prep kits are within the scope ofthe application, and are partially described by Slatko et al. in“Overview of next-generation sequencing technologies.” Current protocolsin molecular biology 122.1 (2018): e59. Current sequencing methods maybe referred to herein as Next Generation Sequencing (NGS). NGS includesmany sequencing by synthesis technologies, including technologies basedon clonal expansion (e.g., bridge amplification in Illumina sequencing)and single molecule sequencing technologies.

Sequencing Library Preparation and Applications

As used herein, library preparation generally refers to preparation of asample for sequencing. Resulting polynucleotides may have sequencingadapters and may be sample barcoded (indexed).

These and other library preparation methods and kits may be adapted forthe subject invention, and are partially described by Head et al. in“Library construction for next-generation sequencing: Overviews andchallenges” Biotechniques. 2014; 56(2): 61-passim. and described furtherherein.

Of note, sample preparation steps prior to library preparation arewithin the scope of the application, and include without limitation oneor more of sample lysis, nucleic acid purification, and enrichment of aparticular group of nucleic acids (such as genomic DNA (gDNA), RNA,mRNA, target mRNA, etc.). Sequencing may be of RNA or gDNA targets, suchas in whole genome sequencing, whole transcriptome sequencing, targetspecific sequencing such as TCR/BCR sequencing, or chromatinaccessibility sequencing.

Library preparation steps may include fragmentation, reversetranscription (e.g., with tailing and template switching), and additionof sequencing adaptors and/or sample barcodes (e.g., through PCR)

Additional steps include harvesting from a microfluidic device, poolingbased on sample quantitation (sample normalization), depletion steps(such as of ribosomal RNA through enzymatic degradation, cleavage,hybridization, etc.), cleanup steps to remove unwanted artefacts such asshort products (e.g., primer dimer), amplification of pooled sample(e.g., with p5/P7 primers), and quantification of pooled sample prior tosequencing.

Fragmentation and Library Prep

In general, the core steps in preparing RNA or DNA for NGS analysis are:(i) fragmenting and/or sizing the target sequences to a desired length,(ii) converting target to double-stranded DNA, (iii) attachingoligonucleotide adapters to the ends of target fragments, and (iv)quantitating the final library product for sequencing.

Fragmentation may be performed by heating, shearing, or enzymatically(e.g., with a DNAse, RNAase, restriction enzyme, transposase, etc.).

The size of the target DNA fragments in the final library is a keyparameter for NGS library construction. Three approaches are availableto fragment nucleic acid chains: physical, enzymatic, and chemical. DNAfragmentation is typically done by physical methods (i.e., acousticshearing and sonication) or enzymatic methods (i.e., non-specificendonuclease cocktails and transposase tagmentation reactions). In ourlaboratory, acoustic shearing with a Covaris instrument (Covaris,Woburn, Mass.) is typically done to obtain DNA fragments in the 100-5000bp range, while Covaris g-TUBEs are employed for the 6-20 Kbp rangenecessary for mate-pair libraries. Enzymatic methods include digestionby DNase I or Fragmentase, a two enzyme mix (New England Biolabs,Ipswich Mass.).

However, Fragmentase produced a greater number of artificial indelscompared with the physical methods. An alternative enzymatic method forfragmenting DNA is Illumina's Nextera tagmentation technology (Illumina,San Diego, Calif.) in which a transposase enzyme simultaneouslyfragments and inserts adapter sequences into dsDNA. This method hasseveral advantages, including reduced sample handling and preparationtime.

Desired library size is determined by the desired insert size (referringto the library portion between the adapter sequences), because thelength of the adaptor sequences is a constant. In turn, optimal insertsize is determined by the limitations of the NGS instrumentation and bythe specific sequencing application. For example, when using Illuminatechnology, optimal insert size is impacted by the process of clustergeneration in which libraries are denatured, diluted and distributed onthe two-dimensional surface of the flow-cell and then amplified. Whileshorter products amplify more efficiently than longer products, longerlibrary inserts generate larger, more diffuse clusters than shortinserts. Optimal library size is also dictated by the sequencingapplication. For exome sequencing, more than 80% of human exomes areunder 200 bases in length

In the case of microRNA (miRNA)/small RNA library preparation, thedesired product is only 20-30 bases larger than the 120 bp adaptordimers. Therefore, it is critical to perform a gel size selection toenrich the libraries as much as possible for the desired product.

Library preparation from DNA samples for sequencing whole genomes,targeted regions within genomes (for example exome sequencing), ChIP-seqexperiments, or PCR amplicons (see below) follows the same generalworkflow. Ultimately, for any application, the goal is to make thelibraries as complex as possible (see below).

Numerous kits for making sequencing libraries from DNA are availablecommercially from a variety of vendors. Competition has driven pricessteadily down and quality up. Kits are available for making librariesfrom microgram down to picogram quantities of starting material.However, one should keep in mind the general principle that morestarting material means less amplification and thus better librarycomplexity.

With the exception of Illumina's Nextera prep, library preparation mayinclude one or more of: (i) fragmentation, (ii) end-repair, (iii)phosphorylation of the 5′ prime ends, (iv) A-tailing of the 3′ ends tofacilitate ligation to sequencing adapters, (v) ligation of adapters,and (vi) some number of PCR cycles to enrich for product that hasadapters ligated to both ends. The primary differences in an Ion Torrentworkflow are the use of blunt-end ligation to different adaptersequences.

Takara's sample prep kits implement first strand synthesis, tailing andtemplate switching. A tailed first strand is synthesized from a firstprimer (e.g., poly(T), target specific, or degenerate primer). Anoligonucleotide with a 3′ sequence complementary to the tail sequenceand provides a template primer binding site incorporated into the firststrand by extension. Another primer binding site may have been providedby the first primer, or can be added by PCR with another primer (e.g.,poly(T), target specific, or degenerate primer). The incorporated primerbinding sites can be used for subsequent PCR and incorporation ofadaptors sequences (e.g., indexes, read sequences, amplificationsequences, etc.).

An oligonucleotide is hybridize Amplification is performed with a primerto the tail sequence and an application specific primer, such as arandomer (N6) primer, target specific primer, poly(A) primer. Sites foramplification by primers (including adaptor sequences) are introduced by

Once the starting DNA has been fragmented, the fragment ends may beblunted and 5′ phosphorylated using a mixture of three enzymes: T4polynucleotide kinase, T4 DNA polymerase, and Klenow Large Fragment.Next, the 3′ ends are A-tailed using either Taq polymerase or KlenowFragment (exo-). Taq is more efficient at A-tailing, but Klenow (exo-)can be used for applications where heating is not desired, such aspreparing mate-pair libraries. During the adapter ligation reaction theoptimal adapter:fragment ratio is ˜10:1, calculated on the basis of copynumber or molarity. Too much adapter favours formation of adapter dimersthat can be difficult to separate and dominate in the subsequent PCRamplification. Bead or column-based clean-ups can be performed after endrepair and A-tail reactions, but after ligation we find bead-basedclean-ups are more effective at removing excess adapter dimers.

To facilitate multiplexing, different barcoded adapters can be used witheach sample. Alternatively, barcodes can be introduced at the PCRamplification step by using different barcoded PCR primers to amplifydifferent samples. High quality reagents with barcoded adapters and PCRprimers are readily available in kits from many vendors. However, allthe components of DNA library construction are now well documented, fromadapters to enzymes, and can readily be assembled into “home-brew”library preparation kits.

An alternative method is the Nextera DNA Sample Prep Kit (Illumina),which prepares genomic DNA libraries by using a transposase enzyme tosimultaneously fragment and tag DNA in a single-tube reaction termed“tagmentation”. The engineered enzyme has dual activity; it fragmentsthe DNA and simultaneously adds specific adapters to both ends of thefragments. These adapter sequences are used to amplify the insert DNA byPCR. The PCR reaction also adds index (barcode) sequences. Thepreparation procedure improves on traditional protocols by combining DNAfragmentation, end-repair, and adaptor-ligation into a single step. Thisprotocol is very sensitive to the amount of DNA input compared withmechanical fragmentation methods. In order to obtain transpositionevents separated by the appropriate distances, the ratio of transposasecomplexes to sample DNA is critical. Because the fragment size is alsodependent on the reaction efficiency, all reaction parameters, such astemperatures and reaction time, are critical and must be tightlycontrolled.

RNA Sequencing Library Prep

It is important to consider the primary objective of an RNA sequencingexperiment before making a decision on the best library protocol. If theobjective is discovery of complex and global transcriptional events, thelibrary should capture the entire transcriptome, including coding,noncoding, anti-sense and intergenic RNAs, with as much integrity aspossible. However, in many cases the objective is to study only thecoding mRNA transcripts that are translated into the proteins. Yetanother objective might be to profile only small RNAs, most commonlymiRNA, but also small nucleolar RNA (snoRNA), piwi-interacting RNA(piRNA), small nuclear RNA (snRNA), and transfer RNA (tRNA). While wewill endeavour to describe the principles of RNA sequencing libraries inthis review, it is not possible to explain all of the differentprotocols available. Interested readers should research the many optionsthemselves.

One major limitation in miRNA library construction arises when theamount of input RNA is low (e.g., <200 ng total RNA); short adapterdimers compete in the RT-PCR reaction with the desired product,adapters, and miRNA inserts. When too many adapter dimers are presentthey stream up the gel during the size selection step and contaminatethe product bands.

For mRNA sequencing libraries, methods have been developed based on cDNAsynthesis (reverse transcription) using random primers, oligo-dTprimers, or by attaching adapters to mRNA fragments followed by someform of amplification. mRNA can be primed by random oligomers or by ananchored oligo-dT to generate first strand cDNA. If random priming isused, the rRNA must first be removed or reduced. rRNA can be removedusing oligonucleotide probe-based reagents, such as Ribo-Zero(Epicenter, Madison, Wis.) and RiboMinus (Life Technologies, Carlsbad,Calif.). Alternatively, poly-adenylated RNA can be positively selectedusing oligo-dT beads. Such poly-A tail may be added by end repair (e.g.,A-tailing enzyme) to enable capture of short or fragmented RNA.Alternatively or in addition, a bead may comprise oligonucleotides thatspecifically hybridize to one or more target nucleic acids, such as TCRand/or BCR sequences. Alternatively or in addition, a bead may compriseoligonucleotides that specifically hybridize to one or more targetnucleic acids, such as TCR and/or BCR sequences. Alternatively or inaddition, target specific probes may hybridize to one or more targetnucleic acids, such as TCR and/or BCR sequences, and said probes maycomprise binding moieties that enable specific binding by beads.

It is often desirable to create libraries that retain the strandorientation of the original RNA targets. For example, in some casestranscription creates anti-sense RNA constructs that may play a role inregulating gene expression. In fact, long noncoding RNA (IncRNA)analysis depends on directional RNA sequencing. Methods for preparingdirectional RNA-seq libraries are readily available. The concept is toperform the cDNA reaction and remove one of the two strands selectively,by incorporating dUTP into the second strand cDNA synthesis reaction.The uracil-containing strand can then be removed enzymatically (NEBNextUltra Directional RNA Library Prep Kit for Illumina) or prevented fromfurther amplification with a PCR polymerase that cannot recognize uracilin the template strand (Illumina TruSeq Stranded Total RNA kit). Inaddition, actinomycin D is frequently added to the first strand cDNAsynthesis reaction to reduce spurious antisense synthesis during thefirst strand synthesis reaction.

An alternative and hybrid method utilizes random or anchored oligo-dTprimers with an adapter sequence on the 5′ end of the primer to initiatefirst strand cDNA synthesis. Next, in a procedure called templateswitching (shown in FIG. 4B), a 3′ adapter sequence is added to the cDNAmolecule. This method has a distinct advantage in that the first strandcDNA molecule can be PCR amplified directly without second strandsynthesis using the unique sequence tag put on the 3′ end by thetemplate switching reaction. A 5′ unique sequence tag is also introducedby standard priming in the first strand synthesis.

Targeted DNA Sequencing

Targeted sequencing allows investigators to study a selected set ofgenes or specific genomic elements; for example, CpG islands andpromoter/enhancer regions. A common application of targeted sequencingis exome sequencing and high quality kits are commercially available;SureSelect (Agilent Technologies), SeqCap (Roche NimbleGen, Madison,Wis.) and TruSeq Exome Enrichment Kit (Illumina). All three capturemethods are based on probe hybridization to enrich sequencing librariesmade from whole genome samples. Life Technologies has commercialized analternative approach based on highly multiplexed, PCR-based AmpliSeqtechnology. There are options to customize all these products andinvestigators can design capture or PCR probes for target regionscovering from thousands to millions of bases within a genome.

Hybridization capture approaches generally work well but can suffer fromoff-target capture and struggle to effectively capture sequences withhigh levels of repetition or low complexity (i.e., the HumanHistocompatibility Locus region). The PCR-based AmpliSeq method is moreefficient with lower amounts of DNA. It should also be noted that probesare based on a reference sequence, and variations that substantiallydeviate from the reference, as well as significant insertion/deletionmutations, are not always going to be identified.

Sequencing of short amplicons also makes obtaining entire sequencespossible in either a single read or using a paired-end read design.Here, adapters can be added directly to the ends of the amplicons andsequenced to retain haplotype information essential for reconstructingantibody or T cell receptor gene sequences as well as identifyingspecies in micro-biome projects.

However, it is often necessary to design longer amplicons for targetedsequencing applications. In this case, the PCR products need to befragmented for sequencing. Amplicons can be fragmented as-is usingacoustic shearing, sonication, or enzymatic digestion. Alternatively,they can be first concatenated into longer fragments using ligationfollowed by fragmentation. One problem associated with ampliconsequencing is the presence of chimeric amplicons generated during PCR byPCR-mediated recombination. This problem is exacerbated in lowcomplexity libraries and by overamplification. The presence of the PCRprimer sequences or other highly conserved sequences presents atechnical limitation on some sequencing platforms that utilizefluorescent detection (i.e., Illumina). This can occur withamplicon-based sequencing such as microbiome studies using 16S rRNA forspecies identification. In this situation, the PCR primer sequences atthe beginning of the read will generate the exact same base with eachcycle of sequencing, creating problems for the signal detection hardwareand software. This limitation is not an issue with Ion Torrent systems(not fluorescence-based) and can be addressed on Illumina systems bysequencing multiple different amplicons in the same lane wheneverpossible. An alternative strategy we employ is to use several PCRprimers during PCR of a specific amplicon. Each primer has a differentnumber of bases (typically 1-3 random bases) added to the 5′ end tooffset/stagger the order of sequencing when adapters are ligated to theamplicons.

Additional Sequencing Approaches

Originally developed as a low-throughput PCR-based assay, theintroduction of NGS technology has allowed ChIP-seq to be efficientlyapplied on a genome wide scale. The general principle of this assayinvolves immunoprecipitation of specific proteins along with theirassociated DNA. The procedure usually requires DNA-protein crosslinkingwith formaldehyde followed by fragmentation of the chromatin usingmicro-coccal nuclease (MNase) and/or sonication. Specific antibodies areused to target the protein or histone modification of interest, at whichpoint the DNA is purified and subjected to high throughput sequencing.The sequencing results should be compared with a proper control. Datafrom a successful ChIP-seq should be enriched for the sequences thatwere crosslinked to the targeted protein/modified histone.

RNA binding proteins (RBPs) recognize ribonucleic acid motifs includingspecific sequences, single-stranded backbones, secondary structures, anddouble-stranded RNA (72,73). These interactions involve all types ofRNAs and occur at every step from transcription to degradation. Manysteps in the post-transcriptional processing of messenger RNA overlap,resulting in multiple RBP complexes bound to a transcript at any givenmoment in its existence. RIP-seq can be done with protein-specificantibodies or by expressing tagged versions of the RBPs of interest.Furthermore, RIP-seq provides the ability to characterize the functionof an RBP in a specific cell type and/or cell state based on thepopulation of bound RNAs.

Methylation of the 5 position of cytosine (5mC) is the most common formof DNA methylation, with 60%-80% of the 28 million CpG dinucleotides inthe human genome being methylated. While genome-wide hypomethylation hasbeen linked to increased rates of mutation and chromosomal instability,hypermethylation of promoters inhibits gene transcription. DNAmethylation is also essential for genetic imprinting, suppression oftransposable elements, and X chromosome inactivation. Aberrant DNAmethylation is associated with many diseases including cancer,autoimmune diseases, inflammatory diseases, and metabolic disorders.Methylation sensitive restriction enzyme sequencing (MRE-seq) relies onrestriction enzymes that are sensitive to CpG methylation. Affinityenrichment of methylated DNA requires either antibodies specific formethylated DNA (MeDIP) or other proteins capable of binding methylatedDNA (MBDseq). Treatment of DNA with sodium bisulfite results in thechemical conversion of unmethylated cytosine to uracil while methylatedcytosines are protected.

ATAC-seq identifies accessible DNA regions by probing open chromatinwith hyperactive mutant Tn5 Transposase that inserts sequencing adaptersinto open regions of the genome. While naturally occurring transposaseshave a low level of activity, ATAC-seq employs the mutated hyperactivetransposase. In a process called “tagmentation”, Tn5 transposase cleavesand tags double-stranded DNA with sequencing adaptors. The tagged DNAfragments are then purified, PCR-amplified, and sequenced usingnext-generation sequencing. Sequencing reads can then be used to inferregions of increased accessibility as well as to map regions oftranscription factor binding sites and nucleosome positions. The numberof reads for a region correlate with how open that chromatin is, atsingle nucleotide resolution.

Addition of Adaptors and Sample Indexes

As described herein, sequences may be added to polynucleotides of thesample through transposition, ligation, tailing and template switching,and/or PCR with primers (e.g., that are degenerate, target specific, orthat hybridize sequences added to fragments by transposition, ligationor tailing and template switching). Added sequences may flank inserts,such as cDNA or gDNA inserts. Added sequences may provide primer bindingsites for further amplification, sequencing adaptors, unimolecularidentifiers, and/or sample indexes (barcodes) that allow for pooling ofsamples. A sequencing adaptor may include indexes and optional indexprimers, amplification elements (e.g. for bridge amplification duringsequencing), and read primers for sequencing.

Additional Steps

Additional steps include harvesting from a microfluidic device,depletion steps (such as of ribosomal RNA through enzymatic degradation,cleavage, hybridization, etc.), sample normalization, and pooling priorto sequencing. Sample normalization, and in particular, samplenormalization based on suppression qPCR, is described further herein.

Bias

The main objective when preparing a sequencing library is to create aslittle bias as possible. Bias can be defined as the systematicdistortion of data due to the experimental design. Since it isimpossible to eliminate all sources of experimental bias, the beststrategies are: (i) know where bias occurs and take all practical stepsto minimize it and (ii) pay attention to experimental design so that thesources of bias that cannot be eliminated have a minimal impact on thefinal analysis.

The complexity of an NGS library can reflect the amount of bias createdby a given experimental design. In terms of library complexity, theideal is a highly complex library that reflects with high fidelity theoriginal complexity of the source material. The technological challengeis that any amount of amplification can reduce this fidelity. Librarycomplexity can be measured by the number or percentage of duplicatereads that are present in the sequencing data. Duplicate reads aregenerally defined as reads that are exactly identical or have the exactsame start positions when aligned to a reference sequence. One caveat isthat the frequency of duplicate reads that occur by chance (andrepresent truly independent sampling from the original sample source)increases with increasing depth of sequencing. Thus, it is critical tounderstand under what conditions duplicate read rates represent anaccurate measure of library complexity.

Using duplicate read rates as a measure of library complexity works wellwhen doing genomic DNA sequencing, because the nucleic acid sequences inthe starting pool are roughly in equimolar ratios. However, RNA-seq isconsiderably more complex, because by definition the starting pool ofsequences represents a complex mix of different numbers of mRNAtranscripts reflecting the biology of differential expression. In thecase of ChIP-seq the complexity is created by both the differentialaffinity of target proteins for specific DNA sequences (i.e., highversus low). These biologically significant differences mean that thenumber of sequences ending up in the final pool are not equimolar.

However, the point is the same—the goal in preparing a library is toprepare it in such a way as to maximize complexity and minimize PCR orother amplification-based clonal bias. This is a significant challengefor libraries with low input, such as with many ChIP-seq experiments orRNA/DNA samples derived from a limited number of cells. It is nowtechnologically possible to perform genomic DNA and RNA sequencing fromsingle cells. The key point is that the level of extensive amplificationrequired creates bias in the form of preferential amplification ofdifferent sequences, and this bias remains a serious issue in theanalysis of the resulting data. One approach to address the challenge isa method of digital sequencing that uses multiple combinations ofindexed adapters to enable the differentiation of biological andPCR-derived duplicate reads in RNA-seq applications (41,42). A versionof this method is now commercially available as a kit from BiooScientific (Austin, Tex.).

When preparing libraries for NGS sequencing, it is also critical to giveconsideration to the mitigation of batch effects. It is also importantto acknowledge the impact of systematic bias resulting from themolecular manipulations required to generate NGS data; for example, thebias introduced by sequence-dependent differences in adaptor ligationefficiencies in miRNA-seq library preparations. Batch effects can resultfrom variability in day-to-day sample processing, such as reactionconditions, reagent batches, pipetting accuracy, and even differenttechnicians. Additionally, batch effects may be observed betweensequencing runs and between different lanes on an Illumina flow-cell.Mitigating batch affects can be fairly simple or quite complex. When indoubt, consulting a statistician during the experimental design processcan save an enormous amount of wasted money and time.

There are many ways to minimize bias during library preparation. Withina single experiment, we aim to start with samples of similar quality andquantity. We also use master mixes of reagents whenever possible. Oneparticularly egregious source of bias is from amplification reactionssuch as PCR; it is well documented that GC content has a substantialimpact on PCR amplification efficiency. PCR enzymes such as Kapa HiFi(Kapa Biosystems, Wilmington, Mass.) or AccuPrime Taq DNA PolymeraseHigh Fidelity (Life Technologies) have been shown to minimizeamplification bias resulting from extremes of GC content.

In addition to enzymatic steps, bias can be reduced in purificationsteps by pooling barcoded samples before gel or bead purification. Inthe case of miRNA-seq libraries, we first run the individual librarieson an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif.) toquantitate the miRNA peaks. We use this information to create barcodedlibrary pools of up to 24 samples and then perform gel purification in asingle lane of an agarose gel to avoid sizing variation between samples.

Sample Normalization

Sample normalization as referred to herein is a physical step in whichpolynucleotides of samples to be pooled are quantified to determine theamount of each sample to add to the pool in order to achieve uniformsequencing across samples. This process may also called libraryquantification and pooling. Quantification is typically performed bytraditional qPCR, or mobility (e.g., electrophoresis) assay such as on aBioanalyzer (a chip-based capillary electrophoresis system), to quantifythe amount of a desired product (polynucleotide) across samples. Samplesare then pooled based on this quantitation, e.g., so as to improveuniformity of read depth across samples. For example, samples with alower concentration of a library prepped polynucleotide (or desiredlibrary prepped polynucleotide) may be added to the pool at greatervolume. Library prepped polynucleotides may be sample barcoded (e.g.,comprise dual indexes), and may or may not include additional adaptorsequences. Read depth can be defined as the number of reads in a givensequencing run, and can further be defined as number of successful reads(e.g., reads mapped to a known sequence or genome). A related concept,sequencing depth uniformity, may also be used. The desired product maybe, for example, a library prepped polynucleotide having an insert of agiven length.

Traditional quantification methods may amplify artefacts such as primerdimer, or by mobility based assays such as electrophoresis in whichartefacts such as bubble DNA (formed from adaptor rehybridization withinsert mismatch) runs at similar speed as desired long products. Assuch, an aspect of the subject application is the use of suppression PCR(e.g., suppression qPCR) to quantify long desirable products overartefacts such as primer dimer, bubble DNA, and products with shortinserts.

Read-depth is directly proportional to sequencing costs and thus lessvariation leads to better results and less costly sequencing.

Suppression PCR

Suppression PCR been used to enrich for long products, as short productsform hairpins at the primer binding site, for example to increase longproducts for use as vectors or to enrich for long products that forsequencing. Suppression PCR for regulation of product length wasdescribed by Shagin et al. in “Regulation of average length of complexPCR product.” Nucleic Acids Research 27.18 (1999): e23-i. To theinventor's knowledge, suppression qPCR and its use in librarynormalization has not been disclosed. Suppression PCR is illustrated inFIG. 5.

In suppression PCR as used herein, a single primer (or primers)complementary to an inverted repeat will preferentially extend longerproducts in which the inverted repeat is spaced farther apart, as thoselonger products tend to remain linear. Shorter products, for example, inwhich the inverted repeat is spaced apart by less than 100, less than80, less than 50, or less than 30 nucleotides, tend to form a hairpin inwhich the inverted terminal repeat forms the neck and prevents primerhybridization.

As described above, quantification methods may amplify artefacts fromlibrary preparation (e.g., shown in FIG. 6) such as primer dimer thatinterferes with qPCR, or bubble DNA which interferes with mobility basedassays such as capillary electrophoresis. As such, an aspect of thesubject application is the use of suppression PCR (e.g., suppressionqPCR) to quantify long desirable products over artefacts such as primerdimer, bubble DNA, and products with short inserts.

In certain aspects, suppression PCR is done with a primer that includesa sequence identical to at least 6, at least 8, at least 10, at least12, or at least 15 nucleotides of a spaced inverted repeat (andtherefore complementary to that length of nucleotides of the otherspaced inverted repeat sequence). Sample preparation may be with primeridentical to an inverted repeat of a polynucleotide (e.g., andcomplementary to the partner inverted repeat, such that it hybridizes toat least one inverted repeat during PCR).

Quantitation by suppression PCR may direct pooling of samples tonormalize for read uniformity. In certain aspects, aliquots fromsamples, but not polynucleotides in the sample pool itself, undergosuppression PCR.

Suppression PCR may enrich for longer amplicons. For example,suppression PCR may enrich for long polynucleotides (e.g., with morethan 100, more than 150, more than 200, more than 300, or more than 400nucleotides between inverted repeats) at least 5 fold (e.g., at least 10fold, at least 25 fold, at least 50 fold, at least 80 fold, at least 100fold) more than shorter polynucleotides (e.g., with less than 100nucleotides, less than 80 nucleotides, less than 50 nucleotides, lessthan 30 nucleotides, or less than 20 nucleotides between invertedrepeats).

Suppression PCR may preferentially amplify longer polynucleotides. Forexample, suppression PCR may amplify long polynucleotides (e.g., withmore than 100, more than 150, more than 200, more than 300, or more than400 nucleotides between inverted repeats) at a PCR cycle efficiency ofat least 0.20 (e.g., 0.25, 0.3, 0.4, 0.5, or 0.6) more than for shorteramplicons (e.g., with less than 100 nucleotides, less than 80nucleotides, less than 50 nucleotides, less than 30 nucleotides, or lessthan 20 nucleotides between inverted repeats). For example, the PCRefficiency for a long polynucleotide may be greater than 1.6, 1.75, 1.8,1.85, or 1.9. The PCR efficiency for a short polynucleotide may be lessthan 1.6, 1.5, 1.4, or 1.3.

Quantitation may be by qPCR (i.e., such as suppression qPCR) asdescribed herein. Alternative methods of quantifying suppression PCR arealso described herein. The length of the library quantificationstandards may be between 150 and 800 nucleotides, such as between 200and 600 nucleotides. Methods may further include melting curve analysisof the suppression PCR products. Quantification may be based on adilution series of a library quantification standard.

Some library prep workflows introduce palindromic sequences (spacedinverted repeats), which form hairpins in short products. Longersequences can be quantified by suppression qPCR, in which a singleprimer complementary to an inverted repeat is used. Shorter productsform hairpins, as the inverted repeats are in closer proximity, whichcompetes with the primer and decreases amplification efficiency. If theamplification efficiency of long (e.g., >200 nt) products is 1.8 foldper cycle, and the amplification efficiency of short (e.g., <50 ntinsert) products is around 1.5, then over 24 cycles the long productswill amplify at (1.8{circumflex over ( )}24)/(1.5{circumflex over( )}24)=80 times more than the short products.

Library preparation workflows can lead to issues with read-depthuniformity per sample in each pool on sequencing runs. For example, inthe initial commercial protocol, the Advanta™ RNA-Seq NGS Library PrepKit (Fluidigm Corp) did not include a method to quantify each samplelibrary and normalize prior to pooling. Read-depth is directlyproportional to sequencing costs and thus less variation leads to betterresults and less costly sequencing. We seek to employ a method ofquant-norm prior to pooling that also doesn't require any beadpurification.

Aspects of a kit described herein use random primers, e.g., to introduceIllumina adaptor sequences through PCR, which creates unwanted primerdimer (e.g., p5 and p7 primer) that interferes with quantification bytraditional qPCR. Primer dimer forms to a greater extent with lowerinputs. When sample inputs are of variable quantity and/or quality, anunnormalized (or poorly normalized) pool will result in non-uniformread-depth. Bubble DNA produced by adapter-driven rehybridizationcomplicates quantification by mobility (such as on a gel or bioanalyzerinstrument), as bubble DNA runs at a similar speed to longer doublestranded products that are desired.

Other library prep methods may also create undesired short products thatcomplicate quantification, and that form hairpin structures due. Forexample, short fragments formed from transposases that introduceinverted terminal repeats may create similar issues that can beaddressed by the invention.

As suppression PCR preferentially amplifies long products over shorthairpins, suppression qPCR would allow quantification of such longproducts. Sample normalization by pooling samples based on suppressionqPCR would allow for uniform sequencing of long reads across samples. Assuch, samples with abundant long products would not need to beoversequenced to achieve adequate sequencing depth for other samples inthe pool, even if artefacts prevent or inhibit traditionalquantification methods from being used for sample normalization.

In certain aspects, suppression qPCR may be combined with melting curveanalysis to ensure short products were not amplified to higher abundancethan long products (e.g., when short products vastly outnumber longproducts initially past the point that could be remediated bysuppression qPCR, such as more than 80 fold in the above example).

Of note, other quantification workflows may not be suitable. Primerdimer formed with low inputs interferes with traditional qPCR. When theprimer sequences include the inverted repeat, these primer dimers willform hairpins with a neck defined by the inverted repeat.

Bubble DNA formed by annealing of adapters on different insertsinterferes with mobility based methods (e.g., electrophoresis).

Additional library prep workflows would lend themselves toquantification by suppression qPCR, which would preferentially amplifylonger products that do not form hairpins. For example, inverted repeatsintroduced in transposase based workflows may form hairpins in smallerfragments.

Aspects of sample normalization by suppression qPCR find use in anylibrary prep workflow that pools samples (not tied to Advanta workflowor IFC) and results in unwanted short hairpin by-products that have aknown inverted repeat forming the neck. For example, primer (e.g.,adapter) dimer may form if some samples have low input. Short productsmay form if sample nucleic acids are fragmented (e.g., RNA in FFPEsamples). In either or both cases, if the primers have a shared sequence(such as a palindromic sequence 8 or more, 10 or more, or 12 or morenucleotides in length), these short products will form hairpinstructures, and qPCR with primer(s) that hybridize the palindromicsequence will preferentially amplify long “readworthy” products overshort hairpins that do not present single stranded palindromic sequence.In another example, a transposase based workflow such as for ATAC seq orWGS may incorporate inverted terminal repeats that form hairpinstructures when the flanked sample DNA fragment is short.

PCR conditions (e.g., temperature and/or time of steps in the PCR cycle,number of cycles, buffer, etc.) may be tuned to improve suppression(e.g., the preferential amplification of long polynucleotides overshort). In certain aspects, annealing and/or extension steps may be runat a higher temperature (e.g., at least 3, 5, or 10 degrees Celsiushigher) in later cycles (e.g., starting at a cycle after cycle 1, 2, 5,10, etc.). This may be most beneficial when the primer for suppressionPCR comprises a 5′ sequence that does not hybridize to thepolynucleotide, but that will hybridize to amplicons of earlier cycles(increasing the melting temperature of primer hybridization after thefirst cycle). Short amplicons would still form hairpins at these highertemperatures, as the neck of the hairpin structure could include the 5′sequence.

Sample Polynucleotide

Sample polynucleotides for sample normalization may be any libraryprepped nucleotide that is sample barcoded (e.g., indexed) such thatsamples can be demultiplexed after pooling. The sample polynucleotidemay have sequencing adaptors, or such sequencing adaptors may be addedafter pooling. For sample normalization by suppression qPCR, samplepolynucleotides may have spaced inverted repeats (i.e., inverted repeatsseparated by another sequences, such as an insert).

Polynucleotides may be library prepped for sequencing (e.g., referred toas a sample library), and may comprise adapters (e.g., one or moresequences to assist sequencing, such as indexes, read primer bindingsites, indexing primer binding sites, amplification primer binding sitessuch as P5/P7 sequences). Polynucleotides (e.g., adapter regions ofpolynucleotides) may include spaced inverted repeats as describedherein. Adapters and/or spaced inverted repeats may be flanking aninsert, such as a cDNA or gDNA sequence. The insert may be of variablelength, such as when fragmentation is for sample preparation.Polynucleotides may include sample barcodes, such as on adaptorsequences. Sample barcodes may be dual indexes.

In certain aspects, polynucleotides comprise spaced inverted repeats,such as two spaced inverted repeats. The spaced inverted repeats are atleast 6, at least 8, at least 10, at least 12, or at least 15nucleotides long. Spaced inverted repeats are within 50 nucleotides oftheir respective ends (3′ and 5′ ends). For example, spaced invertedrepeats may be terminal inverted repeats.

Spacing between inverted repeats may be variable. For example, longerpolynucleotides in a sample may have more than 100, more than 150, morethan 200, more than 300, or more than 400 nucleotides between invertedrepeats. Shorter polynucleotides may have less than 100 nucleotides,less than 80 nucleotides, less than 50 nucleotides, less than 30nucleotides, or less than 20 nucleotides between inverted repeats.Aspects of the subject application may include suppression PCR thatpreferentially amplifies longer polynucleotides. Some polynucleotidesmay include an insert, such as a cDNA or gDNA sequence, flanked by theinverted repeats. The insert may be randomly generated, or may be targetspecific (e.g., gene specific) sequence. The insert sequence may be anendogenous sequence or its reverse complement.

Polynucleotides may include, or may be sample prepared to include, aspaced inverted repeat (e.g., of a sequencing adaptor). For example,polynucleotides may include Illumina p5 and p7 sequencing adaptors. Incertain aspects, a sample has an average polynucleotide length of lessthan half of the average polynucleotide length across the samples

Production of Sample Polynucleotide

Sample polynucleotides may be provided by any of the methods describedherein for sample preparation (e.g., library preparation). For example,PCR may incorporate spaced inverted repeats (e.g., in addition to sampleindexes and/or sequencing adaptors). In another example, transposasesmay fragment target DNA and introduce spaced inverted repeats, oftendescribed in the art as “inverted terminal repeats”. An example ofsample production (library preparation), using a microfluidic workflowand sample normalization, is shown in FIG. 7.

Polynucleotides may be library prepped for sequencing (e.g. referred toas a sample library), and may comprise adapters (e.g., one or moresequences to assist sequencing, such as indexes, read primer bindingsites, indexing primer binding sites, amplification primer binding sitessuch as P5/P7 sequences). Polynucleotides (e.g., adapter regions ofpolynucleotides) may include spaced inverted repeats as describedherein. Adapters and/or spaced inverted repeats may be flanking aninsert, such as a cDNA or gDNA sequence. The insert may be of variablelength, such as when fragmentation is for sample preparation.Polynucleotides may include sample barcodes, such as on adaptorsequences. Sample barcodes may be dual indexes.

In certain aspects, polynucleotides comprise spaced inverted repeats,such as two spaced inverted repeats. The spaced inverted repeats are atleast 6, at least 8, at least 10, at least 12, or at least 15nucleotides long. Spaced inverted repeats may be within 80 nucleotides,within 50 nucleotides, within 30 nucleotides, or within 20 nucleotidesof their respective ends (3′ and 5′ ends). For example, spaced invertedrepeats may be terminal inverted repeats.

Spacing between inverted repeats may be variable. For example, longerpolynucleotides in a sample may have more than 100, more than 150, morethan 200, more than 300, or more than 400 nucleotides between invertedrepeats. Shorter polynucleotides may have less than 100 nucleotides,less than 80 nucleotides, less than 50 nucleotides, less than 30nucleotides, or less than 20 nucleotides between inverted repeats.Aspects of the subject application may include suppression PCR thatpreferentially amplifies longer polynucleotides. Some polynucleotidesmay include an insert, such as a cDNA or gDNA sequence, flanked by theinverted repeats. The insert may be randomly generated, or may be targetspecific (e.g., gene specific) sequence. The insert sequence may be anendogenous sequence or its reverse complement.

Polynucleotides may include, or may be sample prepared to include, aspaced inverted repeat (e.g., of a sequencing adaptor). For example,polynucleotides may include Illumina p5 and p7 sequencing adaptors. Incertain aspects, a sample has an average polynucleotide length of lessthan half of the average polynucleotide length across the samples.

Samples may be prepared by steps described in other sections.

Primers for Suppression PCR

A suppression PCR primer may be identical to an inverted repeat (andtherefore complementary to its partner), or may include a subsequencethat is identical or similar enough to specifically hybridize to aninverted repeat (or portion thereof) under stringency conditions of aPCR reaction.

In certain aspects, a primer for suppression PCR may have a 3′ sequenceidentical to an inverted repeat or a portion thereof (i.e.,complementary to the other inverted repeat). The sequence may beidentical to at least 6, at least 8, at least 10, at least 12, or atleast 15 nucleotides of the inverted repeat. The single primer mayselectively amplify longer products (e.g., having more space betweenspaced inverted repeats), and is sufficient to drive the PCR reaction inthe absence of another primer (e.g., in the presence of master mix and asuitable polymerase and thermocycling conditions). When the primerincludes a sequence identical to a spaced inverted repeat at the 3′ endof the primer, it may only hybridize when the polynucleotide is notforming a hairpin with a neck defined by the inverted repeats.

The primer may include a 5′ sequence that is not complementary to thepolynucleotide but that increases suppression of short amplicons overlong amplicons produced in earlier PCR cycles. For example, the 5′sequence may be at least 4, at least 6, at least 8, at least 10, atleast 12, or at least 15 nucleotides. The 5′ sequence may increase thelength of inverted repeats in amplicons, such that hairpins form with alonger neck (increasing suppression of shorter amplicons). As such, PCRannealing and/or extension temperature may, at least in initial cycles,be low (such as less than 60 degrees Celsius, less than 56 degreesCelsius, or less than 52 degrees Celsius). Later cycle annealing and/orextension temperatures may be higher than in initial cycles as describedherein. In general, PCR annealing and/or extension temperature may bemore than 50 and/or less than 75 degrees Celsius.

Suppression PCR and kits thereof may only use one primer, or may use twodifferent primers both having a 3′ sequence identical to at least 6, atleast 8, at least 10, at least 12, or at least 15 nucleotides of aspaced inverted repeat.

In some embodiments, the primer can (e.g., a single primer can), in thepresence of master mix and polymerase, amplify sample polynucleotideshaving inverted repeats spaced 200 or more nucleotides apart at 0.25 orgreater cycle efficiency than sample polynucleotides having invertedrepeats spaced 50 nucleotides or less. The primer may, in the presenceof master mix and polymerase, amplify the library quantificationstandard at 1.8 or greater efficiency per cycle, but will amplify samplepolynucleotides having inverted repeats spaced by 50 nucleotides or lessat 1.5 or less efficiency in per cycle.

The inverted repeat the primer hybridizes to (e.g., or is identical to)may be introduced by sequencing adaptors, such as those used in librarypreparation kits described herein. As such, suppression PCR primers thathybridize an inverted repeat of Illumina adapters provided by librarypreparation kits described herein are within the scope of theapplication.

Quantification

Sample libraries may be quantified to determine the amount of individualsamples to add to a sample pool prior to sequencing. As describedherein, suppression PCR may be used to amplify polynucleotides ofaliquots from samples. Polynucleotides may be library prepped reactionproducts that comprise spaced inverted repeats (e.g., alongside samplebarcode(s) and/or sequencing adaptors) flanking an insert sequence, suchas cDNA or gDNA. Suppression PCR may preferentially amplifypolynucleotides with longer inserts, as shorter polynucleotides maypreferentially form hairpin structures in which the inverted repeatsform a double stranded neck that prevents primer hybridization and/orextension.

Suppression PCR products may be quantified to determine the amount of asample (e.g., sample library) to add to a pool of samples.Quantification may be performed during suppression PCR, such as in asuppression quantitative PCR or “qPCR”. In qPCR, abundance of doublestranded DNA (dsDNA) is measured over multiple cycles using a dyeindicator (such as an dsDNA intercalating dye), and the linear phase ofamplification curve is used to calculate a starting amount of the targetthat was amplified. Other forms of quantitation may include end pointdetection (e.g., measuring amount of amplified target after a set numberof runs, such as by a dye or by detection of amplification products runon a gel) or digital PCR.

Pooling may be performed based on the quantification of the samples. Forexample, quantification may be used to determine which samples to pooland/or how much (e.g., volume) of particular samples to pool. In certainaspects, multiple sample pools may be created.

Metrics of Improvement

The final library pool formed based on suppression qPCR quantificationof the subject methods and/or kits may provide one or more metrics ofsuccess described herein. Some metrics of improvement are shown in FIGS.8 and 9.

In certain aspects, one or more additional or alternative metrics belowmay be used. The library pool may be of at least 50 fmols, 100 fmols,200 fmols, 300 fmols, 500 fmols, or 1000 fmols (e.g., and have at least24 or 48 samples). The final library pool may provide a sequencing readdepth uniformity of more than 80% or more than 90% of samples having atat least half, or at least two thirds, the read depth of the averageacross samples.

Library normalization may read depth uniformity across pooled samples.For example, library normalization described herein may result in atleast a two-fold reduction (e.g., at least a 3-fold reduction, or atleast a 5 fold reduction) in read depth variation as measured bystandard deviation and as compared to no normalization, or as comparedto normalization based on normal qPCR (not suppression qPCR).

In some embodiments, fewer than 5% of the samples are at a read depthless than 50% of the average read depth across samples. For ample, incertain aspects, no library normalized samples are at a read depth lessthan 50% (e.g., less than 40%, less than 25%, or less than 10%) of theaverage read depth across samples. In addition, more than 5% of thesamples may be at a read depth of less than 50% (e.g., less than 40%,less than 25%, or less than 10%) of the average read depth acrosssamples, if the library were not normalized or were normalized by normalqPCR.

In some embodiments, all of normalized samples may have more than 2500genes detected, such as more than 5000 genes detected, more than 7500genes detected or more than 10,000 genes detected. In addition, at leastsome samples may have fewer than 5000 genes detected, fewer than 2500genes detected, fewer than 1000 genes detected if the library were notnormalized (e.g., for the same total number of reads).

Library normalization may result in at least a two-fold reduction (e.g.,at least a 3-fold reduction, or at least a 5 fold reduction) insequencing costs as compared to no normalization or as compared tonormalization based on qPCR quantitation (e.g., after a bead cleanupstep but not by suppression qPCR). In certain aspects, sequencing costis the cost needed to achieve adequate coverage of all samples in thepool.

Kits

Kits of the subject application may include reagents and/or devices forperforming any of the methods described herein, including methods oflibrary preparation and/or normalization. Kits described herein in thecontext of library preparation for sequencing and/or quantitation ornormalization of libraries for sequencing may be adapted with componentsdescribed herein and/or for method steps described herein.

In certain aspects, a sample normalization kit (e.g., libraryquantification kit) may include a DNA standard having spaced invertedrepeats and a single primer the hybridized one of the inverted repeats.The single primer may selectively amplify longer products (e.g., havingmore space between spaced inverted repeats), and is sufficient to drivethe PCR reaction in the absence of another primer (e.g., in the presenceof master mix and a suitable polymerase and thermocycling conditions).

In certain aspects, a library prep kit may include reagents to addinverted repeats, sample indexes, and optionally sequencing adaptors tosample polynucleotides. The kit may further include and a single primerthe hybridized one of the inverted repeats. The single primer mayselectively amplify longer products (e.g., having more space betweenspaced inverted repeats), and is sufficient to drive the PCR reaction inthe absence of another primer (e.g., in the presence of master mix and asuitable polymerase and thermocycling conditions).

A kit may further include components for determining pooling of samplesbased on quantification, such as a workbook (e.g. spreadsheet) thattakes qPCR measurement input and outputs instructions for pooling ofquantified samples, such as which samples to pool and/or how much (e.g.,volume) of particular samples to pool.

Alternative uses of Suppression PCR

In certain aspects, suppression PCR may be used for applications outsideof library quantification.

In some embodiments, primers may comprise sequences (e.g., internal orat their 5′ ends) that are identical of one another (e.g., and more than6, 8 or 12, or 15 nucleotides in length), and may comprise 3′ sequencesthat are different from one another and that hybridize targetnucleotide. The identical sequence can introduce spaced inverted repeatsduring amplification such that short products (e.g., primer dimer) formhairpins and are not efficiently amplified in future cycles, such asduring qPCR or dPCR. The temperature of annealing and/or extension maybe increased (e.g., by at least 3, 5 or 10 degrees Celsius, starting ata cycle after cycle 1, 2, 3, 5, 10, etc.) such that the 3′ sequence nolonger hybridizes the original target nor a short amplicon (e.g., primerdimer) that form hairpins.

In certain aspects, the 3′ sequences is degenerate (e.g., a randomer ofmore than 3, 4, 6, or 8 nucleotides). In multiple cycle amplificationwith randomer primers, amplicons of subsequent amplification cycles canget successively smaller as new randomer primers hybridize new sites,which is undesirable for many applications (including sequencing).However, the formation of hairpin structures in short amplicons producedwith inverted repeat introducing primers may promote amplification oflong amplicons in future cycles. Further, an additional primer orprimers (e.g., having the identical sequence from the first two primersat the 3′ end and an adaptor sequence) can amplify long amplicons thatdo not form hairpins. The additional primer may be in excess of theearlier described primer(s).

Microfluidic Devices

Microfluidic devices described herein refer to devices that processfluid volumes (e.g., sample volumes) on the microliter scale (e.g., oneul to hundreds of ul) or less, such as 0.1 nl to 100 ul, 1 nl to 10 ul,5 nl to 1 ul, or 10 nl to 100 nl. Alternatively, microfluidic devicesrefer to fluidic devices with channels, chambers or other fluidicarchitectures with a dimension on the micrometer scale (e.g., one um tohundreds of um) or less, such as 100 nm to 1 mm, or 1 um to 100 um. Thearchitecture of microfluidic devices of the subject application mayallow for controlled loading, isolation, mixing and/or harvesting ofsample, reagents, and solutions. Microfluidic devices may parallelizesample preparation to the point that sample normalization(quantification and pooling) prior to sequencing is of great benefit. Anexemplary microfluidic device is shown in FIGS. 1 and 2, and exemplarymicrofluidic architecture is shown in FIGS. 3 and 4.

Polynucleotides may be produced in a multi-step reaction in amicrofluidic device, such as for sequencing library preparation. Themicrofluidic device may be, without limitation, an elastomericmicrofluidic device or a positive displacement liquid handler. Nucleicacid may be enriched on the microfluidic device using beads, forexample, RNA may be enriched by poly-A capture. Nucleic acid may befragmented, reverse transcribed, and/or sample barcoded by PCR in themicrofluidic device. Sample polynucleotides produced in the microfluidicdevice may include spaced inverted repeats.

Enrichment

Enrichment mechanisms include immobilization of biomolecules, such assample nucleic acids, on a solid support within the microfluidic device.The solid support may be a fluid permeable matrix, a wall of a channelor chamber, or beads as described further herein.

Bead retention mechanisms may be based at least partially on particlecontact with any suitable physical barrier(s) disposed in a microfluidicnetwork. Such particle-barrier contact generally restricts longitudinalparticle movement along the direction of fluid flow, producingflow-assisted retention. Flow-assisted particle-barrier contact also mayrestrict side-to-side/orthogonal (transverse) movement. Suitablephysical barriers may be formed by protrusions that extend inward fromany portion of a channel or other passage (that is, walls, roof, and/orfloor). For example, the protrusions may be fixed and/or movable,including columns, posts, blocks, bumps, walls, and/orpartially/completely closed valves, among others. Some physicalbarriers, such as valves, may be movable or regulatable. Alternatively,or in addition, a physical barrier may be defined by a recess(es) formedin a channel or other passage, or by a fluid-permeable membrane. Otherphysical barriers may be formed based on the cross-sectional dimensionsof passages. For example, size-selective channels may retain particlesthat are too large to enter the channels. A sieve architecture mayprovide a plurality of openings through which fluid may flow but beadslarger than the hole may be retained.

Chemical retention mechanisms may retain particles based on chemicalinteractions. The chemical interactions may be covalent and/ornoncovalent interactions, including ionic, electrostatic, hydrophobic,van der Waals, and/or metal coordination interactions, among others.Chemical interactions may retain particles selectively and/ornonselectively. Selective and nonselective retention may be based onspecific and/or nonspecific chemical interactions between particles andpassage surfaces.

Such retention mechanisms may be part of a column that retains beads forenrichment of biomolecules in a unit cell.

Beads

Beads may be manufactured from inorganic materials, or materials thatare synthesized chemically, enzymatically and/or biologically.Furthermore, beads may have any suitable porosity and may be formed as asolid or as a gel. Suitable bead compositions may include plastics(e.g., polystyrene), dextrans, glass, ceramics, sol-gels, elastomers,silicon, metals, and/or biopolymers (proteins, nucleic acids, etc.).Beads may have any suitable particle diameter or range of diameters.Accordingly, beads may be a substantially uniform population with anarrow range of diameters, or beads may be a heterogeneous populationwith a broad range of diameters, or two or more distinct diameters.

Beads may be associated with any suitable materials. The materials mayinclude compounds, polymers, complexes, mixtures, phages, viruses,and/or cells, among others. For example, the beads may be associatedwith a member of a specific binding pair, such as a receptor, a ligand,a nucleic acid, a member of a compound library, an affinity reagent(such as and antibody, avidin/biotin, or a derivative thereof), and/orso on. For example, beads may be functionalized with streptavidin tobind to biotinylated molecules. In another example, beads arefunctionalized with (i.e., present on their surface) a chemical groupsuch as carboxy functional groups (e.g., to bind nucleic acid). Inanother example, beads are functionalized with an oligonucleotide thatbinds nucleic acid in the sample, such as through hybridization to apoly-A sequence or target specific sequence(s). In certain aspects,beads may be functionalized with an affinity reagent, such as anantibody (e.g., or fragment thereof), aptamer, tetramer (e.g., such asan MHC or MHC-peptide), receptor (e.g., a T-cell receptor), an avidin(e.g., streptavidin) or biotin. For example, beads may be functionalizedwith antibodies to one or more protein targets, such as peptide/proteinbiomarkers or viral antigen, as described further herein. In certainaspects, beads may be functionalized with a pathogen or antigen thereof,such as a viral particle or viral antigen as described further herein.In certain aspects, beads may be functionalized with an oligonucleotide,such as a ssDNA that specifically hybridizes to a target nucleotidesequence (e.g., a specific RNA, cDNA, or gDNA sequence). In certainaspects, beads functionalized to bind different target samplebiomolecules may be used in admixture for a multiplexed assay.

Beads may be magnetic to allow for ease of enrichment and/or washing intube. Alternatively, beads may be not be magnetic (such as when they areretained through physical barriers on a microfluidic device).

Microfluidic Devices for Sample Preparation and Detection

Aspects of the methods described herein may be performed on amicrofluidic device (or a fluidic device), which are themselves withinthe scope of the subject application. An exemplary microfluidic deviceis shown in FIG. 2.

Sample processing may be performed at least in part on a microfluidicdevice. For example, a plurality of samples may be processed in parallelon a microfluidic device (e.g., in separate unit cells), harvested, andthen pooled for sequencing. The device may process at least 2, at least4, at least 12, at least 24, at least 48, at least 96, or at least 384different samples. Processing on the microfluidic device may includelibrary preparation, such as formation of polynucleotide reactionproducts having sequencing adaptors and/or sample indexes. Depending onthe application, sample processing on a microfluidic device may includeone or more of biomolecule (e.g., nucleic acid) enrichment, washing,elution, fragmentation, reverse transcription (when the biomolecule isRNA), and PCR (e.g., to incorporate sequencing adaptors and/or samplebarcodes such as dual indexes). Additional steps, such as cleanup,amplification, quantification for sample normalization, and/or poolingmay be performed off the microfluidic device or in a downstream fluidicdevice.

Microfluidic devices may comprise a network of flow channels and/or mayinclude a microliter or nanoliter scale pipetting apparatus (such as apositive displacement pipetting arm) to perform multistep reactions in amultiwell plate.

In certain aspects, enrichment may be performed on the microfluidicdevice. For example, the microfluidic device (e.g., a unit cell of themicrofluidic device) may include a column for immobilization of targetbiomolecules on a solid support. In certain aspects, the column mayinclude beads or may be configured to retain beads, and can be describedas a specialized chamber. For example, beads in a column may be packedupstream of a sieve, such that the beads may be retained under flow offluid (e.g., sample, wash solution, reagents, etc.). The beads may befunctionalized with a chemical group or biomolecule for binding targetbiomolecules, as described further herein. Beads may be loaded into thecolumn of the microfluidic device, after which sample (and optionally awash solution) may be flowed across the beads. Alternatively, beads maybe mixed with sample, and optionally washed, prior to injection into themicrofluidic device. This alternative may improve enrichment and/orreduce sample loading time at the cost of less automation on themicrofluidic device. In either case, the microfluidic device allows forbead-based enrichment to increase the amount of biomolecules processedin a unit cell (while maintaining a low amount of reagent used andallowing for parallel processing of samples in a single microfluidicdevice).

The microfluidic device may provide one or more sample processing sites(e.g., downstream of a column in a unit cell). For example, the devicemay provide at least 2, at least 3, at least 4, or at least 5 processingsites in a unit cell. The sample processing sites may be fluidicallyisolated from one another and/or the column during operation, such asthrough valves positioned along the unit cell, until a mixing stepbetween one or more processing sites and/or the column is initiated.Mixing may be by interface free mixing or active mixing such as dilationpumping or peristaltic pumping.

The microfluidic device may enable loading of different reagents intodifferent processing sites (e.g., prior to a mixing step). Themicrofluidic device may have one or more waste channel to remove excesssolution, such as solution suspending beads packed into the column. Awaste channel, reagent channels, and/or sample channels may share aportion of their length with one another.

Sample channels may be configured to inject sample (e.g., beads) into afirst junction of a unit cell (e.g., proximal to a column). Sample maybe flowed through the column and out a waste outlet to allow loading ofbeads and/or to pass sample biomolecules over beads in the column.Different samples may be delivered to different unit cells.

A plurality of reagent channels may be configured to introduce reagentsinto different sample processing sites (such as chambers of differentsample processing site). Such reagents may perform any of the sampleprocessing steps described throughout this application. At least some(e.g., or all) reagent channels may share a portion of their channellength along a shared channel, and the microfluidic device may beoperated to flow a given reagent through the shared channel at differenttimes. For example, the microfluidic device may include a multiplexorthat may be operated to control which reagent inlet is used to load aprocessing site of the unit cell. The shared channel may deliver reagentthrough a second junction of the unit cell. Reagent flowed through theshared channel may be directed to different sample processing sites(e.g., via a network of valves). Alternatively, at least some reagentchannels may each deliver a reagent directly to a specific sampleprocessing site (e.g., and not through a shared channel). In certainaspects, reagent loading of multiple unit cells may be performedsimultaneously and/or identically. Loading of reagents into sampleprocessing sites may be by blind filling (e.g., where one end of thechamber or channel of the sample processing site is blocked by a closedvalve or wall, or by flow of the reagent through the sample processingsite and out a waste outlet.

The microfluidic device may deliver solutions to the unit cell,including wash and/or elution solutions that are flowed through thecolumn after loading the sample, and harvest solution that is used toremove prepared sample from the device.

The microfluidic device may have additional channels and junctions forintroduction and removal of such solutions. For example, a waste outletchannel may collect excess sample, reagent, and/or solutions. A harvestoutlet may collect prepared sample. In certain aspects, a harvestsolution may be flowed from a harvest inlet, through the unit cell, andinto the sample inlet (which functions as a harvest outlet for eachsample. In general, sample, reagents, solution and waste channels mayshare channel segments and/or junctions of entry to the unit cell, whendoing so simplifies architecture and does not interfere with the sampleprocessing reactions or result in contamination.

In one example, samples may be lysed and nucleic acids (e.g., RNA) maybe immobilized on beads prior to loading the beads in a column of amicrofluidic device for sample preparation. The microfluidic device maybe operated to perform reverse transcription, and library preparation(including sample indexing). Library prepped samples may be harvestedfrom the microfluidic device and then pooled. In certain aspects,pooling of the library prepped samples may be based on a samplenormalization, such a suppression qPCR as described herein.

As described herein, the microfluidic device may include a plurality ofvalves. The valves and channels of the microfluidic device may bearranged to load sample into the column, direct different reagents (fromdifferent reagent inlets) into separate sample processing sites (e.g.,chambers), isolate reaction sites, and mix fluids between reaction sites(e.g., by circulating fluids across sample processing loops).

The microfluidic device may be operated by a system, such as acontroller described herein. Such as system may also comprise athermocycler for driving reactions, such as reverse transcription and/orPCR. In certain aspects, the microfluidic device may be an elastomericmicrofluidic device with elastomeric valves as described herein.

Microfluidic devices of the subject application may perform any numberof assays, including but not limited to: PCR (such as endpoint PCR,digital PCR (dPCR), or quantitative PCR (qPCR)), immuno-PCR (such asimmunoqPCR), proximity assay, Elisa, reverse transcription,premaplification (e.g., targeted, multiplexed targeted, or not-specificpreamplification such as whole genome or whole transcriptomeamplification), sample encoding/indexing, and so forth. In certainaspects, the method of detection is qPCR (i.e., real-time PCR) in whicha reaction is interrogated across a number of thermal cycles such thatan abundance of the target can be determined. qPCR provides a a cyclethreshold (CT) that relates to the abundance of the target. qPCR methodson array microfluidic devices are described in US patent publicationnumber US20160153026, which is incorporated herein by reference. Thecycle threshold in qPCR is discussed at length in US patent publicationnumber US20080129736 which is incorporated herein by reference.

In proximity ligation described in US patent publication number20050003361, binding moieties are provided on proximity probes thathybridize to a splint template and are ligated. However, the bindingmoieties are for coupling of each probe to an affinity reagent (e.g., anantibody) and the splint template is a synthetic target (e.g., asynthetic single stranded DNA sequence) that enables ligation of theprobes when their affinity reagents are bound in proximity. Proximityassays, such as proximity extension, are also described in the contextof microfluidic devices in US patent publication number US20160024557,which is incorporated herein by reference. Proximity assays to detectprotein targets may be performed alongside detection of RNA or DNAtargets in the same sample as described further herein.

Elastomeric Microfluidic Devices

Suitable microfluidic devices include elastomeric microfluidic deviceswith elastomeric valves. Such elastomeric valves may be pressurized todeflect an elastomeric membrane into a flow channel of the microfluidicdevice, thereby controlling fluidic communication. Backpressure ofinlets can drive fluids (e.g., samples, reagents, solutions, etc.)through channels that are not blocked off by closed valves.

Early disclosure of elastomeric microfluidic devices can be found inU.S. Pat. No. 7,601,270, disclosure of loop channels and peristalticpumps can be found in U.S. Pat. No. 7,351,376, disclosure of dead end(blind) filling can be found in U.S. Pat. No. 7,766,055, disclosure ofsurface functionalization and immobilization can be found in U.S. Pat.No. 7,691,333, disclosure of multiplexor architecture can be found inU.S. Pat. No. 7,691,333, and disclosure in multi-step processingarchitecture can be found in U.S. Pat. No. 9,429,500, all of which areincorporated by reference herein.

In the context of elastomeric microfluidic devices:

A “flow channel” refers generally to a flow path through which asolution can flow.

The term “valve” unless otherwise indicated refers to a configuration inwhich a flow channel and a control channel intersect and are separatedby an elastomeric membrane that can be deflected into or retracted fromthe flow channel in response to an actuation force.

An “isolated reaction site” generally refers to a reaction site that isnot in fluid communication with other reactions sites present on thedevice. When used with respect to a blind channel, the isolated reactionsite is the region at the end of the blind channel that can be blockedoff by a valve associated with the blind channel.

The term “elastomer” and “elastomeric” has its general meaning as usedin the art. Thus, for example, Allcock et al. (Contemporary PolymerChemistry, 2nd Ed.) describes elastomers in general as polymers existingat a temperature between their glass transition temperature andliquefaction temperature. Elastomeric materials exhibit elasticproperties because the polymer chains readily undergo torsional motionto permit uncoiling of the backbone chains in response to a force, withthe backbone chains recoiling to assume the prior shape in the absenceof the force. In general, elastomers deform when force is applied, butthen return to their original shape when the force is removed. Theelasticity exhibited by elastomeric materials can be characterized by aYoung's modulus. The elastomeric materials utilized in the microfluidicdevices disclosed herein typically have a Young's modulus of betweenabout 10 Pa-100 GPa, in still other instances between about 20 Pa-1 GPa,in yet other instances between about 50 Pa-10 MPa, and in certaininstances between about 100 Pa-1 MPa. Elastomeric materials having aYoung's modulus outside of these ranges can also be utilized dependingupon the needs of a particular application.

Systems for Operating Microfluidic Devices

A system coupled to the microfluidic device may include controllers offluid flow in the microfluidic device, such as a pneumatic controller.Alternatively or in addition, the same may include a thermocyclers fordriving reactions such as lysis, nucleic acid purification, reversetranscription, and PCR in reaction sites (e.g., sample processing sites)of the microfluidic device. Disclosure of microfluidic carriers,controllers and thermocycler interfaces can be found in U.S. Pat. No.7,704,735, which is incorporated herein by reference.

Exemplary Library Preparation and Normalization Workflow

An exemplary RNA sequencing library preparation workflow is describedbelow, aspects of which may be performed by the subject methods, devicesand/or kits.

This exemplary method includes the following steps:

-   -   i) Preparing and loading RNA and reagents on a fluidic circuit,        then oligo d(T) beads.    -   ii) Library preparation is performed on the Fluidigm 48.Atlas        IFC (integrated fluidic circuit)    -   iii) Harvested barcoded libraries are normalized (quantified by        qPCR and pooled)    -   iv) The pooled library is cleaned up (with Agencourt AMPure XP        beads), amplified by PCR using sequencing adaptors (primers that        amplify from the P5 and P7 portions of the adaptor sequences),        and the pooled samples are quantified by qPCR prior to        sequencing.

Library preparation of step ii) can include the below steps:

-   -   Poly(a) RNA capture on solid-phase beads    -   Elute and fragment poly(A) RNA    -   Reverse-transcribe and template-switch    -   Sample-barcoding PCR    -   Harvesting of sample libraries

Quantification of barcoded libraries in step iii) can be performedaccording to the below workflow:

-   -   Provide A KAPA Library Quantification Kit (master mix and DNA        standard) modified with a qPCR Primer Premix, and library        dilution buffer (10 mM Tris-HCI, pH 8.0 with 0.05% Tween 20).    -   Dilute the Sample Libraries 50-Fold and aliquot 2 ul of        individual samples    -   Run qPCR on aliquots of the samples using the modified library        quantification kit    -   Import qPCR results into a Normalization Workbook    -   Pool sample libraries according to the Normalization Workbook        output

Splinted Ligation for Target RNA Detection

Small reaction volumes may benefit from advances in sample enrichmentworkflows. In addition, variability in sample input and quality may leadto variable sequencing depth across pooled samples, such that the samplepool must be over-sequenced to obtain a suitable depth for poor samples,markedly increasing cost. A common solution is to quantify desiredlibrary products and normalize the amount of sample libraries added tothe pool based on this quantification. Such quantification methodsinclude traditional qPCR and mobility based methods that detect libraryproducts based on length. However, artefacts from library preparationworkflows can interfere with existing methods of quantification.Improved methods of quantification may result in a multiple foldreduction in sequencing cost.

Aspects of parallel processing herein include methods and kits forsplinted ligation based detection of a target nucleic acid, such as atarget RNA. Splinted ligation aspects described herein may obviate theneed for reverse transcription and optionally pre-amplification beforedetection (e.g., detection by PCR based detection such as qPCR, ordetection by sequencing).

Splinted ligation has been reported by Maroney et al. in “A rapid,quantitative assay for direct detection of microRNAs and other smallRNAs using splinted ligation” Rna 13.6 (2007): 930-936. Maroney reportedRNA detection in which the target RNA is not the splint template andsubsequent detection of the ligation product by gel electrophoresis.Splinted ligation detection by PCR has been reported by Blewett et al.in “A quantitative assay for measuring mRNA decapping by splintedligation reverse transcription polymerase chain reaction: qSL-RT-PCR”Rna 17.3 (2011): 535-543. However, in the method reported by Blewett,the target RNA was not the splint template and the splinted ligationproduct required reverse transcription prior to PCR based detection. Inproximity ligation described in US patent publication number20050003361, binding moieties are provided on proximity probes thathybridize to a splint template and are ligated. However, the bindingmoieties are for coupling of each probe to an affinity reagent (e.g., anantibody) and the splint template is a synthetic target (e.g., asynthetic single stranded DNA sequence) that enables ligation of theprobes when their affinity reagents are bound in proximity. Proximityassays, such as proximity extension, are also described in the contextof microfluidic devices in US patent publication number US20160024557,which is incorporated herein by reference. Proximity assays to detectprotein targets may be performed alongside detection of RNA or DNAtargets in the same sample as described further herein.

None of the above publications provide methods where an endogenousnucleic acid target (e.g., an endogenous target RNA such as a genomicviral RNA or a mammalian gene transcript) in the splint template for twosynthetic splint ligation probes (e.g., DNA or RNA based), therebyobviating need for reverse transcription. Further, none of the abovepublications disclose capture (e.g., for enrichment) of splint ligationprobes on a solid support (e.g., beads) that specifically binds abinding moiety present on one or both of the probes. Still further, noneof the above publications recite one or more splint ligation probes witha sample barcode for selective amplification by a sample barcode primer(e.g., that hybridizes to the sample barcode sequence or reversecomplement), allowing for pooling of samples prior to detection by PCR.One or more of these distinct aspects, optionally in combination with amicrofluidic device and workflow described herein, may provide uniquebenefit. Such aspects, and additional aspects, are discussed furtherherein for any suitable combination in a kit or method.

The subject splinted ligation methods and kits may offer one or moredistinct advantages described below. By using the RNA target as thesplint template, a reverse transcription enzymatic step may be avoided.Such a step may be inhibited in a lysate, blood, saliva, or other fluidsample. Ligase may not be inhibited, or may be less inhibited, is such asample. Further, risk of amplicon contamination may be reduced as thereis no need for off-IFC reverse transcription and preamplification (e.g.,reverse transcription may be unnecessary and preamplification, if neededafter the optional enrichment described below, may be performed on theIFC). Capture (e.g., enrichment) of probes comprising a binding moietyas described herein may allow for separation of the hybridizationproduct from inhibitory components of the lysate, blood, saliva or otherfluid sample prior to ligation. The hybridization scheme describedherein may have a minimal footprint. For example, both probes maytogether hybridize a sequence of the target RNA that is less than 60nucleotides, less than 50 nucleotides, less than 40 nucleotides, or lessthan 30 nucleotides in length. As such, a short or degraded RNA may bedetected by this approach, where a traditional PCR of a larger segmentor poly-A based enrichment and/or reverse transcription, would not besuitable. Such RNA may have been degraded by fixation (such as in FFPEtissue) or in the bodily fluid sample (such as degradation of viral RNAin saliva). In microfluidic (e.g., IFC) based workflows, capture on themicrofluidic device as described herein may provide enrichment of thetarget RNA, hybridization product, and/or ligation product in thereaction sites of the IFC. Such reaction sites may have volume(s) lessthan 1 ul, less than 500 nl, less than 200 nl, less than 100 nl, lessthan 50 nl, or less than 20 nl. As formation of a ligation productrequires binding of two probes adjacent to one another, this methodprovides high specificity. Sample barcoded probes allow sample to bepooled prior to steps such as ligation and/or preamplification to allowuniform sample handling and increase parallel sample processing.Individual samples may be interrogated by sequencing ligation product inpooled samples or by separating the pooled sample into separate reactionvolumes and performing sample specific PCR (e.g., qPCR) using one ormore sample barcode primers as described herein. For example, pooledsample may be split into different reaction volumes (e.g., reactionsites on an array IFC) and target RNA from individual samples may bedetected by using different sample barcode primers to amplify theligation product from different samples in different reaction sites.Such sample barcoded primers may be input through assay inlets of anarray IFC as described herein, and the pooled sample may be inputthrough a sample inlet and optionally captured and/or pre-processed onthe microfluidic device upstream of the array. Detection on an array IFCmay enable parallel sample processing, automation and uniform sampleprocessing, as well as small reaction volumes which results in lowerreagent costs.

Splinted Ligation with Target Nucleic Acid Splint Template

In certain aspects, the target nucleic acid may be an endogenous DNA orRNA. Endogenous target RNA may be viral RNA (e.g., genomic viral RNA) orof a mammalian species, such a gene transcript or non-coding RNA from ahuman, non-human primate, or rodent subject. Samples could be celllysates (e.g., from cell culture or from tissue), cell-free nucleicacids (e.g., from a bodily fluid), or purified nucleic acids, e.g., asdescribed further below.

For example, the target RNA may be a genomic viral RNA, such as of arespiratory virus (e.g., syncytial virus, influenza virus, parainfluenzavirus, metapneumovirus, rhinovirus, and coronavirus). In such cases, asample from a subject (e.g., a human) may be taken to determine viralinfection. The sample may be saliva, nasal swab, blood, or an extractedcomponent thereof. In certain aspects, the viral RNA may be partiallydegraded (such as in a saliva sample) and difficult to detect bytraditional reverse transcription and PCR amplification.

In another example, the target RNA may be an endogenous mammalian (e.g.,rodent, non-human primate, or human) RNA. The mammalian RNA may be agene transcript or a non-coding RNA, such as ribosomal RNAs (rRNAs), aswell as small RNAs such as microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs,exRNAs, scaRNAs, or ncRNAs. In certain aspects, the RNA may befragmented, such as by FFPE fixation and/or long term storage. As such,viral infection and/or strain, and optionally further viral load, may bedetected using splinted ligation methods and/or kits described herein.

While detection of RNA may typically require RNA extraction, reversetranscription and/or preamplification (e.g., of viral cDNA), the subjectsplinted ligation embodiments described herein may obviate one or moreof these steps, thus reducing assay cost and/or increasingparallelization of sample analysis. As such, genotyping or geneexpression may be detected using splinted ligation methods and/or kitsdescribed herein.

In the subject splinted ligation methods and kits, the target RNA actsas a splint. As a target RNA of interest is usually known (e.g., basedon a sequenced, and publically available, viral or mammalian genome ortranscriptome), splint ligation probes (simply referred to as “probes”in the context of splint ligation herein) may be designed tospecifically hybridize the target RNA adjacent to one another such thata 3′-OH end of a first probe is next to a 5′-PO4 end of the secondprobe. Such hybridization forms a splint hybridization product (simplyreferred to as a “hybridization product” in the context of splintligation herein), and can be described as having a nick between the3′-OH and 5′-PO4 ends of the first and second probe respectively.

Splint ligation probes may be DNA or RNA based. DNA based probes mayallow PCR amplification. Probes may each hybridize to a an adjacentsequence on the target nucleic acid, such as a sequence between 10 and30 nucleotides long, between 15 and 25 nucleotides long, less than 40nucleotides, less than 30 nucleotides long, less than 25 nucleotideslong, less than 20 nucleotides long, or less than 15 nucleotides long. Aprobe may have a binding moiety (e.g., attached to the end that is notligated). Such a probe may have a cleavage site to enable separationfrom a solid support (e.g., before preamplification and/or detection bysequencing or PCR). Alternatively or in addition, a probe may have asample barcode for selective amplification of ligation products formedin a specific sample, as described further herein.

Ligation of said probes in the hybridization product may be by asuitable ligase, and forms a splint ligation product (simply referred toas a “ligation product” in the context of splint ligation herein). Suchligation may be by any suitable ligase. For example, if the probes areDNA probes, a ligase such as T4 ligase or PBCV-1 ligase, which has beenshown to ligated nicked DNA on DNA-RNA hybrids by Lohman et al. in“Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNAligase” Nucleic acids research 42.3 (2014): 1831-1844.

The target nucleic acid may be DNA or RNA. Further, the splint ligationprobes may comprise DNA and/or RNA. As such, the hybridization productmay be a DNA-DNA, RNA-RNA, or DNA-RNA hybrid. Ligases suitable for thehybridization product may be chosen by one of skill in the art.

FIGS. 10A and 10B shows an exemplary splint hybridization product andsplint ligation product respectively. FIG. 10A shows an exemplary splinthybridization product of the subject application in which the targetnucleic acid (e.g., a DNA or RNA, such as an endogenous mammalian orviral RNA) acts as the splint template. Two probes specificallyhybridize to adjacent sequences of the target nucleic acid, such that a3′-OH end of the first probe is adjacent to a 5′-PO4 end of the secondprobe. One or both of the probes may further have a barcode sequence,such as a sample barcode. One or both of the probes may have a bindingmoiety (e.g., to allow for capture on a solid support such as beads,such as for enrichment and/or purification). Such capture may be of thehybridization product, followed by ligation of the capturedhybridization product. Alternatively, such capture may be of theligation product formed upon ligation in solution. FIG. 10B shows anexemplary splint ligation product formed from ligation of the two probesat the adjacent region (i.e., nick). The target nucleic acid may stillbe hybridized to the ligation product, or may be degraded (or allowed todegrade, such as degradation of target RNA by heat, RNAse, or anysuitable means). The probes may be DNA or RNA. For example, DNA probesmay allow for subsequent PCR of the ligation product.

FIG. 11 shows exemplary splinted ligation workflows. In all suchmethods, a hybridization product is formed by hybridization of twoprobes to a target nucleic acid (e.g., target RNA) that acts as a splinttemplate. Ligation of the probes in the hybridization product forms aligation product. Detection of the target nucleic acid may be bysequencing of the ligation product sequence, or by PCR (e.g., qPCR) ofthe ligation product as shown in FIG. 11. In certain aspects, such asshown in the leftmost workflow, the hybridization product is ligated andthen detected by PCR without capture or pooling. In certain aspects,such as shown in the middle-left workflow, hybridization product isligated and then captured on a solid support (e.g., enriched and/orpurified) prior to detection by PCR. Of note, the capture step may beperformed before the ligation step, such as when ligation is performedwhen the hybridization product is still bound to a solid support. Incertain aspects, such as shown in the middle, middle-right, andrightmost workflow, at least one probe may have a sample barcodeallowing hybridization products or ligation products to be pooled. Thepool may be separated (e.g., after steps such as ligation, capture,and/or preamplification) and ligation production from different samplesmay be detected in different reaction volumes using sample barcodeprimers. For example, hybridization products may be captured, thenpooled, and then ligated prior to detection by PCR as shown in themiddle workflow. In another example, hybridization products may beligated, then pooled, and then captured prior to PCR as shown in themiddle-right workflow. In another example, hybridization products may bepooled, then captured, and then ligated prior to PCR as shown in theright workflow. In certain aspects, capture, ligation, preamplification,splitting of a pool, and/or PCR detection may be performed in on amicrofluidic device, such as a device comprising a column and/or arrayIFC. For example, capture of hybridization products or ligation productsmay be on beads that are then flowed into a sieve architecture on amicrofluidic device to form a column, or capture may be by flowinghybridization products or ligation products over beads already loaded ina column of the microfluidic device. Detection may be on an array IFC,such as when ligation products are formed for different targets and/orin different samples, as described further herein.

In certain aspects, ligation and preamplification may be performed inthe same reaction step or reaction volume, such as on a microfluidicdevice or in tube.

Splinted Ligation Methods and Kits

The subject application includes the following aspects:

1. A method of library normalization comprising:

-   -   a. obtaining aliquots from a plurality of samples, wherein the        samples comprise polynucleotides comprising spaced inverted        repeats;    -   b. performing suppression PCR on the aliquots of step a;    -   c. quantifying amplification products from step b;    -   d. pooling the plurality of samples to form a library normalized        based on the quantification of step c; and        -   wherein the pooled plurality of samples have not undergone            the suppression PCR of step b.

2. The method of aspect 1, wherein the plurality of samples comprise atleast 8 samples.

3. The method of aspect 1, wherein the samples are library prepped forsequencing.

4. The method of aspect 1, wherein the sample is derived from fixedtissue.

5. The method of aspect 1, wherein suppression PCR is with a primercomprising a sequence identical to at least 8 nucleotides of a spacedinverted repeat.

6. The method of aspect 1 or 5, wherein the sequence identical to thespaced inverted repeat is at the 3′ end of the primer.

7. The method of aspect 6, wherein the primer comprises a 5′ sequencethat is not complementary to the polynucleotide but that increasessuppression of short amplicons over long amplicons produced in earlierPCR cycles.

8. The method of aspect 7, wherein the 5′ sequence is at least 6nucleotides long.

9. The method of aspect 1, wherein suppression PCR is with only oneprimer.

10. The method of aspect 1, wherein the samples are pooled to normalizefor read uniformity.

11. The method of aspect 1, wherein the pooled plurality of samples havenot undergone suppression PCR

12. The method of aspect 1, wherein the suppression PCR enriches foramplicons with more than 200 nucleotides between inverted repeats atleast 25 fold more than amplicons shorter than 50 nucleotides betweeninverted repeats.

13. The method of aspect 1, wherein suppression PCR is with only oneprimer.

14. The method of aspect 1, wherein suppression PCR is with twodifferent primers both comprising a 3′ sequence identical to at least 8nucleotides of a spaced inverted repeat.

15. The method of aspect 1, wherein quantification is by qPCR.

16. The method of aspect 1, wherein quantification is based on adilution series of a library quantification standard

17. The method of aspect 16, wherein the length of the libraryquantification standards is between 150 and 800 nucleotides.

18. The method of aspect 1 or 15, further comprising melting curveanalysis of the amplification products from step b.

19. The method of aspect 1 or 3, wherein the polynucleotides comprisecDNA.

20. The method of aspect 1 or 3, wherein the polynucleotides comprisegDNA.

21. The method of aspect 1 or 3, wherein polynucleotides comprise samplebarcodes.

22. The method of aspect 21, wherein the polynucleotides comprise dualindexes.

23. The method of aspect 1, wherein polynucleotides comprises spacedinverted repeats flanking an insert of variable length.

24. The method of aspect 1, wherein the insert is cDNA.

24. The method of aspect 1, wherein the polynucleotides comprise exactlytwo spaced inverted repeats of length 8 nucleotides or more.

25. The method of aspect 1 or 24, wherein the spaced inverted repeatsare within 50 nucleotides of their respective ends.

26. The method of aspect 25, wherein the spaced inverted repeats areterminal.

27. The method of aspect 1, wherein the spacing of the inverted repeatsis variable.

28. The method of aspect 1, wherein the polynucleotides comprise asequencing adaptor comprising a spaced inverted repeat.

29. The method of aspect 1 or 28, wherein the polynucleotides of thesample comprise Illumina p5 and p7 sequencing adaptors.

30. The method of aspect 28, wherein the spaced inverted repeats are atleast 8 nucleotides long.

31. The method of aspect 1, wherein at least one sample has an averagepolynucleotide length of less than half of the average polynucleotidelength across the samples.

32. The method of aspect 1, further comprising producing thepolynucleotides prior to step a.

33. The method of aspect 32, wherein producing the polynucleotidescomprises 3′ enrichment.

34. The method of aspect 32, wherein producing the polynucleotidescomprises bead based enrichment of the polynucleotides.

35. The method of aspect 32, wherein producing the polynucleotidescomprises reverse transcription.

36. The method of aspect 32 or 35, wherein producing the polynucleotidescomprises tailing and template switching.

37. The method of aspect 32 or 33, wherein producing the polynucleotidescomprises fragmentation.

38. The method of aspect 32, wherein producing the polynucleotidescomprises random priming.

39. The method of aspect 32, wherein producing the polynucleotidescomprises ligation that adds spaced inverted repeats to thepolynucleotide.

40. The method of aspect 32, wherein producing the polynucleotidescomprises PCR introduction of sample barcode.

41. The method of aspect 32 or 40, wherein producing the polynucleotidescomprises PCR introduction of spaced inverted repeats.

42. The method of aspect 32, wherein the polynucleotides are producedfrom RNA.

43. The method of aspect 32, wherein the polynucleotides are producedfrom gDNA.

44. The method of aspect 32, wherein the polynucleotides are produced ina multi-step reaction in a microfluidic device.

45. The method of aspect 44, wherein the microfluidic device is anelastomeric microfluidic device.

46. The method of aspect 44 or 45, wherein nucleic acid is enriched onthe microfluidic device using beads.

47. The method of aspect 46, wherein RNA is enriched by poly-A capture.

48. The method of aspect 47, wherein poly(A) RNA is fragmented, reversetranscribed, and resulting cDNA is sample barcoded by PCR in themicrofluidic device

49. The method of aspect 44, wherein the polynucleotides are produced ina multi-step reaction performed by a positive displacement liquidhandler.

50. The method of aspect 1, further comprising sequencingpolynucleotides of the pooled plurality of samples.

51. The method of aspect 50, wherein sequencing is whole genomesequencing, whole transcriptome sequencing, target specific sequencing,or chromatin accessibility sequencing.

52. The method of aspect 50, wherein the library normalization improvesread depth uniformity across pooled samples.

53. The method of aspect 50, wherein library normalization results in atleast a two-fold reduction in read depth variation as compared tonormalization based on qPCR quantitation but not suppression qPCR.

54. The method of aspect 50, wherein library normalization results in atleast a two-fold reduction in sequencing cost as compared tonormalization based on qPCR quantitation but not suppression qPCR.

55. The method of aspect 50, wherein sequencing cost is the cost neededto achieve adequate read depth of all samples in the pool.

56. The method of aspect 50, wherein adequate read depth is at least5000 reads.

57. The method of aspect 50, fewer than 5% of the samples are at a readdepth less than 50% of the average read depth across samples.

58. The method of aspect 57, wherein no samples are at a read depth lessthan 50% of the average read depth across samples

59. The method of aspect 50, wherein more than 5% of the samples wouldbe at a read depth of less than 50% of the average read depth acrosssamples, if the library were not normalized.

60. The method of aspect 59, wherein at least some samples would be at aread depth of less than 25% of the average read depth across samples, ifthe library were not normalized.

61. The method of aspect 60, wherein all samples in the normalizedlibrary have more than 5000 genes detected.

62. The method of aspect 61, wherein at least some samples would havefewer than 2500 genes detected if the library were not normalized

63. A kit for library quantification of polynucleotides by suppressionqPCR, the kit comprising:

-   -   a library quantification standard comprising spaced inverted        repeats separated by at least 150 nucleotides; and    -   a primer comprising a sequence identical to at least 8        nucleotides of one of the inverted repeats.

64. The kit of aspect 63, wherein the primer can, in the presence ofmaster mix and polymerase, amplify the library quantification standardat 1.75 or greater efficiency per cycle, but will amplify samplepolynucleotides comprising inverted repeats spaced by 50 nucleotides orless at 1.5 or less efficiency in per cycle.

65. The kit of aspect 64, wherein the primer can, in the presence ofmaster mix and polymerase, amplify polynucleotides comprising invertedrepeats spaced 200 or more nucleotides apart at 0.25 or greater cycleefficiency than polynucleotides comprising inverted repeats spaced 50nucleotides or less.

66. The kit of aspect 63, wherein the library quantification standardcomprises sequencing adaptors that comprise the inverted repeats.

67. The kit of aspect 63, wherein the kit further comprise adaptors thatintroduce spaced inverted repeats.

68. A kit for library preparation and quantification, comprising:

-   -   adaptors together providing inverted repeats at least 8        nucleotides in length; and    -   a primer comprising a sequence identical to at least 8        nucleotides of an inverted repeat.

69. The kit of aspect 68, wherein the primer can, in the presence ofmaster mix and polymerase, amplify sample polynucleotides comprisinginverted repeats spaced 200 or more nucleotides apart at 0.25 or greatercycle efficiency than sample polynucleotides comprising inverted repeatsspaced 50 nucleotides or less.

70. The kit of aspect 68, wherein the primer can, in the presence ofmaster mix and polymerase, amplify the library quantification standardat 1.8 or greater efficiency per cycle, but will amplify samplepolynucleotides comprising inverted repeats spaced by 50 nucleotides orless at 1.5 or less efficiency in per cycle.

71. The kit of aspect 63 or 68, wherein the sequence identical to thespaced inverted repeat is at the 3′ end of the primer.

72. The kit of aspect 63 or 68 or 71, wherein the primer comprises a 5′sequence that is not complementary to the polynucleotide but thatincreases suppression of short amplicons over long amplicons produced inearlier PCR cycles.

73. The kit of aspect 72, wherein the 5′ sequence is at least 6nucleotides long.

74. The kit of aspect 63 or 68, wherein the primer is sufficient forsuppression PCR of polynucleotides comprising the spaced invertedrepeats.

75. The kit of aspect 63 or 68, further comprising a different primercomprising a sequence identical to at least 8 nucleotides of a spacedinverted repeat but comprising a different 5′ sequence from the primer.

76. The kit of aspect 68, wherein the adaptors comprise a samplebarcode.

77. The kit of aspect 68 or 76, wherein the adaptors comprise dualindexes. wherein library prepped polynucleotides produced with theadapters comprise spaced inverted repeats

78. The kit of aspect 68, further comprising beads for enriching nucleicacids on a microfluidic device.

79. The kit of aspect 78, wherein oligonucleotides comprising a 3′poly-T sequence are bound to the beads.

80. The kit of aspect 68, 69 or 70, further comprising a microfluidicdevice for bead based enrichment of target nucleotide sequences.

81. The kit of aspect 80, wherein the microfluidic device has a seriesof reaction sites for multi-step sequencing library preparation.

82. The kit of aspect 68 or 80, further comprising reagents for reversetranscription.

83. The kit of aspect 63 or 68, further comprising a passive referencedye

84. The kit of aspect 63 or 68, further comprising a PCR master mix forperforming the qPCR.

85. A kit for performing any one of method aspects 1 to 62.

86. A method comprising library normalization based on suppression qPCR.

87. A method comprising suppression qPCR.

88. A method comprising sequencing a library normalized by suppressionqPCR.

89. A pool of samples normalized based on suppression qPCR of any one ofaspects 1 to 62.

90. A method of processing a splint hybridization product, comprising:

-   -   a) hybridizing a first probe and a second probe to a target        nucleic acid to form a hybridization product, wherein a 3′-OH        end of the first probe is adjacent to a 5′-PO4 end of the second        probe, and wherein at least one of the first probe and the        second probe comprises a binding moiety;    -   b) capturing the hybridization product by specifically binding        the binding moiety to a solid support.

91. The method of aspect 1, wherein the target nucleic acid is a targetRNA.

92. The method of aspect 2, wherein the target RNA is a genomic viralRNA.

93. The method of aspect 3, wherein the target RNA is a mammalian genetranscript.

94. The method of any one of aspects 90 to 93, wherein the solid supportcomprises beads.

95. The method of any one of aspects 90 to 94, wherein the solid supportis on a column of a microfluidic device.

96. The method of any one of aspects 90 to 95, further comprisingligating the hybridization product after capturing the hybridizationproduct.

97. The method aspect 96, further comprising separating the ligationproduct from the solid support.

98. The method of any one of aspects 90 to 95, wherein at least one ofthe first probe and the second probe comprises a sample barcode.

99. The method of aspect 96 or 97, wherein at least one of the firstprobe and the second probe comprises a sample barcode.

100. The method of aspect 99, further comprising combining hybridizationproducts from different samples to form a pool of samples, before orafter step b) of capturing.

101. The method of aspect 100, further comprising detecting targetnucleic acid of different samples by separating the pool of samples intoseparate reaction volumes, and further comprising PCR amplification ofthe ligation product of different samples in different reaction volumesusing sample barcode primers.

102. The method of aspect 101, wherein the PCR amplification is on anarray IFC.

103. The method of any one of aspects 90 to 99, further comprisingdetecting the target nucleic acid by PCR amplification of the ligationproduct.

104. The method of aspect 102 or 103, wherein the PCR amplification isquantitative PCR.

105. The method of aspect 102 or 103, wherein the PCR amplification isendpoint PCR.

106. A method of detecting a splint ligation product, comprising:

-   -   a) hybridizing a first probe and a second probe to a target        nucleic acid to form a hybridization product, wherein a 3′-OH        end of the first probe is adjacent to a 5′-PO4 end of the second        probe;    -   b) ligating the first probe and second probes to form a ligation        product;    -   c) detecting the presence of the ligation product.

107. The method of aspects 106, further comprising capturing thehybridization product or the ligation product on a solid support.

108. The method of aspect 107, wherein the hybridization product iscaptured on the solid support prior to step b) of ligating.

109. The method of aspect 107, wherein the ligation product is capturedon the solid support.

110. The method of any of aspects 107 to 109, wherein the solid supportcomprises beads.

111. The method of aspect 110, wherein the beads are on a microfluidicdevice, or wherein the beads are loaded onto a microfluidic device aftersaid capturing.

112. The method of aspect 111, further comprising PCR amplification ofthe ligation product in the microfluidic device.

113. The method of any of aspects 107 to 112, wherein the first probecomprises a binding moiety on its 5′ end and/or the second probecomprises a binding moiety on its 3′ end.

114. The method of aspect 113, wherein the binding moiety is biotin or aderivative thereof and the solid support comprises avidin orstreptavidin.

115. The method of aspect 114, further comprising separating theligation product from the solid support.

116. The method of aspect 115, wherein separation of the ligationproduct from the solid support is on a microfluidic device, and whereinthe solid support is beads in a column on the microfluidic device.

117. The method of any one of aspects 106 to 116, wherein step c) ofdetecting is by PCR amplification.

118. The method of aspect 117, wherein the PCR amplification is endpointPCR.

119. The method of aspect 117, wherein the PCR amplification isquantitative PCR.

120. The method of any one of aspects 117 to 120, wherein the PCRamplification is on an array IFC.

121. The method of any one of aspects 1 to 116, wherein at least one ofthe first probe and the second probe comprises a sample barcode.

122. The method of aspect 121, further comprising pooling hybridizationproducts or ligation products from different samples.

123. The method of aspect 122, further comprising preamplifying thepooled ligation products by PCR.

124. The method of aspect 122 or 123, further comprising separating thepool into a plurality of reaction sites and detecting ligation productsof different samples in different reaction sites, and wherein step c) ofdetecting comprises using sample barcode primers for PCR amplification.

125. The method of aspect 124, wherein step c) of detection comprisesqPCR using a first primer that hybridizes to the sample barcode and asecond primer that hybridizes target specific sequence or barcode of theligation product, and wherein a plurality of ligation products fromdifferent target RNAs and different samples are detected in separatereaction sites

126. The method of aspect 124 or 125, wherein the PCR amplification isendpoint PCR.

127. The method of aspect 124 or 125, wherein the PCR amplification isquantitative PCR.

128. The method of aspect 122, wherein step c) of detecting is bysequencing of ligation products in the pooled samples.

129. The method of any one of aspects 106 to 128, wherein the targetnucleic acid is a target RNA.

130. The method of aspect 129, wherein the target RNA is a viral RNA.

131. The method of aspect 129, wherein the target RNA is a mammaliangene transcript.

132. The method of any one of aspects 106 to 131, wherein a plurality ofdifferent target nucleic acids are each hybridized by a unique pair oftwo probes in step a) prior to hybridization of each pair of probes toform different ligation products in step b).

133. The method of aspect 132, further comprising detecting ligationproducts from different target nucleic acids by qPCR in differentreaction sites.

134. The method of aspect 133, wherein the different reaction sites areon an array integrated fluidic circuit.

135. The method of aspect 134, further comprising separately detectingligation products from different samples using different sample barcodeprimers in different reaction sites.

136. The method of any one of aspects 106 to 135, further comprisingcapturing hybridization products or ligation products on a solid supportin a microfluidic device.

137. The method of aspect 136, further comprising amplifying theligation product of different samples and/or target RNAs in separatereaction sites in an array of the microfluidic device.

138. The method of any one of aspects 106 to 137, wherein at least oneof the first and second probes are DNA probes.

139. The method of any one of aspects 106 to 138, wherein the targetnucleic acid is RNA and wherein the method does not include reversetranscription of the target RNA.

140. A splint ligation detection kit, comprising:

-   -   a first probe and a second probe that each hybridize to a target        nuclic acid to form a hybridization product,    -   wherein a 3′-OH end of the first probe is adjacent to a 5′-PO4        end of the second probe,    -   wherein the first probe comprises a binding moiety on its 5′ end        and/or the second probe comprises a binding moiety on its 3′        end.

141. The kit of aspect 140, wherein the target nucleic acid is targetRNA.

142. The kit of aspect 141, wherein the target RNA is a viral RNA.

143. The kit of aspect 143, wherein the target RNA is a mammalian genetranscript.

144. The kit of any one of aspects 140 to 143, wherein the bindingmoiety is biotin or a derivative thereof.

145. The kit of any one of aspects 140 to 144, further comprising asolid support that specifically binds the binding moiety.

146. The kit of any one of aspects 140 to 145, further comprising aligase.

147. The kit of any one of aspects 140 to 146, further comprising areagent for separating a captured hybridization product or ligationproduct from the solid support.

148. The kit of any one of aspects 140 to 147, further comprising aplurality of primers that specifically amplify a ligation product underPCR conditions

149. The kit of aspect 148, wherein at least some of the primers aresample barcode primers.

150. The kit of aspect 149, wherein the kit comprises a plurality ofseparated probes that each comprise a different sample barcode.

151. The kit of any one of aspects 140 to 150, wherein the kit comprisesa plurality of probe pairs that each hybridize to different target RNAs,optionally wherein different probe pairs are in mixture.

152. The kit of aspect 151, further comprising target specific primersthat specifically amplify ligation product formed from different targetRNA's, optionally wherein the target specific primers are in mixture.

153. The kit of any one of aspects 140 to 152, further comprising amicrofluidic device comprising a column for bead based enrichment of thehybridization product or a ligation product formed from thehybridization product.

154. The kit of aspect 153, wherein the microfluidic device comprises aseries of reaction sites for multi-step sample processing of ligationproduct.

155. The kit of aspect 153 or 154, wherein the microfluidic devicefurther comprises an array of reaction sites downstream of the series ofreaction sites, wherein each reaction site in the array is configured tomix a different processed sample with reagents from a different assayinlet.

156. The kit of any one of aspects 140 to 152, further comprising anarray IFC.

Capture Based Enrichment

Aspects include capture of splint hybridization products or splintligation products on a solid support. The solid support may includebeads, a column on a microfluidic device (e.g., packed with beads), amatrix, or a planar array. Beads may be any suitable material, e.g., asdescribed elsewhere herein.

The solid support (e.g., beads) may be functionalized to specificallybind a binding moiety of a probe (e.g., incorporated into ahybridization product or ligation product by one or both probes). Incertain aspects, the binding may be by affinity or by covalent binding.For example, the probe may comprise biotin or a derivative thereof, anda bead may comprise avidin or streptavidin (or visa versa). In certainaspects the binding may be covalent, such as through thiol reactivechemistry, amine reactive chemistry, or click chemistry (e.g., betweenTCO and tetrazing, or DBCO and azide). As such, the binding moiety maybe an affinity reagent or analyte, or a covalent binding moiety.Alternatively, a probe may be attached to the bead prior to hybridizingto the target nucleic acid to enable capture of a hybridization producton the bead. Alternatively, a probe may comprise an anchor sequence thathybridizes to an oligonucleotide provided by the solid support (e.g.,the bead).

A probe may have a binding moiety (e.g., attached to the end that is notligated). Such a probe may have a cleavage site to enable separationfrom a solid support (e.g., before preamplification and/or detection bysequencing or PCR).

Bead based capture of hybridization products or ligation products may beperformed “in tube” (i.e., off a microfluidic device such as inindividual tubes or in microwell plates). After such capture, beads maybe flowed into a microfluidic column for additional processing on themicrofluidic device. In certain aspects, beads may be loaded into acolumn on a microfluidic device, and hybridization product or ligationproduct may be flowed through the column to enrich for the product. Themicrofluidic device may comprise a column and downstream sampleprocessing site(s) such as discussed elsewhere herein and shown in FIG.3 or 4. Ligation may occur in tube or, when captured hybridizationproduct is in a microfluidic device, may be performed in themicrofluidic device.

Aspects may include providing bead bound to hybridization product orligation product, as described above. Such product may be separated frombead prior to addition processing (e.g., prior to ligation in the caseof a hybridization product, prior to preamplification, and/or prior toPCR amplification for detection). Separation may be mediated by chemicalor enzymatic cleavage, such as cleavage of dU on the probe by UDG (alsoreferred to as UNG) and/or cleavage by an endonuclease such as APE1.Such a probe may comprise DNA (e.g., other than the dU sequence proximalto a binding moiety). Alternatively or in addition, separation may bemediated by heat. Alternatively, separation may be by displacement, suchas displacement by free biotin of a desthiobiotin binding moiety boundto avidin or streptavidin on the bead (or visa versa). Alternatively orin addition, probes and/or beads may comprise linker (e.g., a PEGlinker) to space the hybridization product or ligation product from thesurface of the bead.

As described further herein, samples may be sample barcoded and pooledbefore or after capture. The above capture methods and reagents may beused for capture for applications outside of splinted ligation, such asapplications described elsewhere herein.

Barcoded Splint Ligation Probes and Pooling

In certain aspects, one or more splint ligation probes may comprise abarcode, such as a sample barcode. Such a probe is shown in thehybridization product of FIG. 10A, and may optionally comprise a bindingmoiety and/or additional components described herein.

The sample barcode may be between 5 and 30 nucleotides, such as between10 and 25 nucleotides, in length. The sample barcode(s) may beincorporated into the ligation product such that it is flanking the siteof hybridization to the target nucleic acid. Barcoded samples (e.g.,barcoded hybridization products or ligation products) may be pooledprior to certain steps, such as ligation, capture, preamplification,and/or detection (e.g., by sequencing or PCR, such as qPCR). Detectionby PCR may involve separating pooled sample (e.g., that has beencaptured, ligated and/or preamplified after pooling) into separatereaction volumes, and separately detecting ligation product fromdifferent samples using sample barcode primers in different reactionvolumes. Sample barcode primers may hybridize to the sample barcode orits reverse complement. In certain aspects, separation may be intoreaction sites of an IFC array, and different sample barcode primers maybe flowed into the array through different assay inlets. Assay specificprimers that bind an assay barcode or target nucleic acid sequence (orits complement) may be used, e.g., in combination with sample barcodeprimers to detect a different combination of a target and sample inseparate reaction sites. Array IFCs and sample barcoding for detectionby PCR is described in US publication number US20100120038, and isincorporated by reference in its entirety. Pooled sample may be preparedin tube and flowed into the array directly from sample inlets, or may beflowed from a unit cell comprising a column and/or processing sites suchas that shown in FIG. 3 or 4.

Sample barcoding and/or pooling may be combined with other aspects, suchas capture, described herein.

Microfluidic Automation and Parallel Processing

FIG. 12 is a schematic of an array integrated fluidic circuit (IFC). Asshown, a plurality of sample inlets 1202A-X provide sample to samplechambers (black boxes). A plurality of assay inlets 1204A-Y provideassay reagents (e.g., primers and optionally additional PCR componentssuch as polymerase, dNTPs and/or cofactors) to assay chambers (whiteboxes). Sample inlets to the array may be directly from wells loadedfrom a user, or may be downstream of a column and/or samplepreprocessing sites as described herein and shown in FIG. 3 or 4. Incertain aspects, assay inlets may provide different target specificprimers.

Such an array may be integrated (in fluidic communication) with unitcells for sample capture and/or processing, such as the unit cells shownin FIG. 3 or 4. Alternatively, sample obtained from a microfluidicdevice shown in FIG. 3 or 4, or prepared in tube off any microfluidicdevice, may be harvested and input to a separate array IFC.

The array IFC may have multiple layers such that sample and assay flowchannels can pass over one another. The array IFC may be elastomeric(e.g., comprise an elastomer such as PDMS), and may further haveelastomeric valves controlled through pressure applied to controlchannels (not shown) to deflect a membrane into a flow channel. Valvesmay be positioned along the dashed flow channels so as to contain sampleand/or assay in their respective chambers (e.g., preventing backwardscontamination after mixing). Valves may be positioned between pairs ofsample an assay chambers to control mixing (e.g., by interface freediffusion). Detailed descriptions of suitable array architecture may befound in US patent publications US20100120038 and US20140193896, both ofwhich are incorporated herein by reference in their entirety.

In certain aspects, assay inlets may provide primers to amplify splintligation products formed as described herein. Alternatively or inaddition, assay inlets may provide sample barcode primers that bindsample barcode sequences (or their reverse complement) on ligationproducts from a specific sample in a pool of samples. Such samplebarcodes may be incorporated by splint ligation probes, and samples maybe pooled before being provided to the array through a sample inlet. Forexample, if 8 samples are pooled for each of 48 different sample inlets,and 8 different assay inlets each amplify ligation product from adifferent sample, then 48×8 (i.e., 386) different samples may be assayedin the array. As such, assay inlets may increase the number of targetsand/or samples detected. As such, an array device may comprise at least12, at least 24, at least 48, or at least 96 separate sample inlets. Inaddition, at least 4, at least 8, at least 12, or at least 24 differentsample barcodes may be flowed through different assay inlets. As such,more samples may be assayed than there are sample inlets. For example,at least 386 different samples may be assayed in the array. The arrayfootprint may be less than 100 square centimetres, such as less than 20square centimetres, or less than 10 square centimetres. In certainaspects, different target specific primers are also flowed intodifferent assay inlets, such that different reaction sites detectdifferent targets from different samples.

Detection of Ligation Products

Detection of splint ligation products may be by sequencing (e.g., ofsample barcode and pooled samples), or by PCR amplification (e.g.,endpoint PCR or quantitative PCR). Methods of library preparation forsequencing are known, and may be performed on a microfluidic device asdescribed herein. Alternatively, PCR products may be quantified todetermine the amount of a sample (e.g., sample library) to add to a poolof samples. Quantification may be performed during PCR, such as in aquantitative PCR or “qPCR”. In qPCR, abundance of double stranded DNA(dsDNA) is measured over multiple cycles using a dye indicator (such asan dsDNA intercalating dye) and the linear phase of amplification curveis used to calculate a starting amount of the target that was amplified.Other forms of quantitation may include end point detection (e.g.,measuring amount of amplified target after a set number of runs, such asby a dye or a target specific probe) or digital PCR.

PCR of ligation product, in tube or on an array IFC, may therefore allowdetection and optionally quantitation of the target nucleic acid. Asdescribed above, one or more probes may have a sample barcode, such thatthe ligation product has a sample barcode on one or both sides. PCRamplification may then be with one or more sample barcode specificprimers, such as to selectively amplify ligation product from a specificsample in a pool of samples as described herein. Alternatively or inaddition, at least some primers may be target specific to allow fordetection of a ligation product from a specific target nucleic acid.

Splinted Ligation Kits

In certain aspects, a kit for parallel sample process may includereagent for splint ligation methods described herein. Such a kit mayhave two splint ligation probes described in any embodiments herein, andmay optionally further comprise ligase for forming ligation products,primers for amplifying ligation products, and/or additional reagents.Splinted ligation kits may further include one or more microfluidicdevices described herein.

Microfluidic Automation and Parallel Processing

A system for controlling fluid flow in the microfluidic device, thermalcontrol, and/or imaging the microfluidic device is described in USpatent publication number US20080088952, which is incorporated herein byreference in its entirety. For example, the system may perform one ormore steps of:

-   -   i) flowing a plurality of samples into reaction chambers of the        microfluidic device;    -   ii) amplifying template nucleic acids from the plurality of        samples; and    -   iii) detecting amplification reactions.

The system may include one or more of:

an automated pressure source for applying a pressure to actuate valvesin an elastomeric microfluidic device and to introduce samples into aplurality of reaction chambers of the elastomeric microfluidic device,wherein the elastomeric microfluidic device includes a carrieraccessible to the automated pressure source;

a thermal platen configured to mate with a portion of the carrier of theelastomeric microfluidic device; and

an optical imaging system comprising a light source, an optical lenssystem, and a detector array camera.

For example, the automated pressure source, the thermal platen, and theoptical imagining system are part of a single platform.

The system may isolate at least some of the plurality of the reactionchambers from one another. The microfluidic device may be any devicedescribed herein, and the system may perform any method describedherein.

In certain aspects, an array microfluidic device (e.g., array IFC) maybe used, such as for detection of splint ligation products (i.e., theirtargets) as described herein. In certain aspects, an array IFC may beintegrated (in fluidic communication) with a unit cell comprising acolumn and/or processing sites as described herein.

FIG. 12 is a schematic of an array integrated fluidic circuit (IFC). Asshown, a plurality of sample inlets 1202A-X provide sample to samplechambers (black boxes). A plurality of assay inlets 1204A-Y provideassay reagents (e.g., primers and optionally additional PCR componentssuch as polymerase, dNTPs and/or cofactors) to assay chambers (whiteboxes). Sample inlets to the array may be directly from wells loadedfrom a user, or may be downstream of a column and/or samplepreprocessing sites as described herein and shown in FIG. 3 or 4. Incertain aspects, assay inlets may provide different target specificprimers.

Integrated Workflows and Microfluidic Devices

As described above, an integrated microfluidic device may thereforeinclude, an array of reaction sites and a plurality of sample processingunit cells comprising a plurality of sample processing sites, whereinthe unit cell is in fluidic communication with a plurality of differentreagent inlets, and wherein sample inlets to the array are downstream ofthe plurality of sample processing sites of the plurality of unit cells.

The plurality of reagent inlets may share a common channel to each unitcell. The microfluidic device may include a multiplexor configured tocontrol which reagent inlet is used to load a processing site of theunit cell.

The plurality of sample processing sites may include a plurality ofloops and/or chambers. Each unit cell further includes one or more of asample inlet channel, a waste outlet channel, additional reagent inlets,and/or additional columns.

Each unit cells may include a plurality of valves configured to controlthe unit cell. The plurality of valves may be configured to deliversample and reagents to different locations in the unit cell. Theplurality of valves may be configured to place sample processinglocations in isolation or in communication with one another. Theplurality of valves may be configured to drive mixing at differentlocations. The plurality of valves are configured to direct flow ofsample or reagents solution out of the unit cell For example, the unitcell includes a peristaltic pump (e.g., defined by a set of valves inseries).

Wherein individual unit cells further includes at least one columnconfigured to retain beads. The column may include a sieve architectureproviding a plurality of openings through which fluid may flow but beadslarger than the hole may be retained. In certain aspects, the unit cellincludes at least two columns, such as columns arranged in series and/orin parallel. For example, the unit cell may include a first column(e.g., for cleanup, such as serum cleanup) fed by a sample/bead inletchannel, and may further include a plurality of columns in parallel(e.g., each fed by a bead inlet and communicating with a plurality ofdownstream sample processing sites), such as is shown in FIG. 17. A unitcell with multiple columns may be used for enrichment of differenttarget biomolecules, as described further herein.

Individual reaction sites of the array of reaction sites may eachinclude an assay chamber and a sample chamber. Sample inlet channels mayprovide sample to the sample chambers and assay inlets provide assayreagents to the assay chambers, for example as shown in FIG. 12.

The microfluidic device may include multiple layers such that sampleinlet flow channels and assay inlet flow channels pass over one another.The microfluidic device is an elastomeric microfluidic device, forexample, may include PDMS (polydimethylsiloxane). Elastomeric valves ofthe device may be defined by the intersection of a flow channel and acontrol channel which are separated by an elastomeric membrane that canbe deflected into or retracted from the flow channel in response to anactuation force.

The microfluidic device includes at least 12 unit cells, at least 24unit cells, at least 48 unit cells, or at least 96 unit cells. The anarray of the microfluidic device may further include at least 3 times,at least 8 times, at least 16 times, or at least 24 times the number ofreactions sites compared to unit cells. For example, each unit cell mayfeed into at least 3, at least 8 k at least 16, or at least 24 differentreaction sites. The different reaction sites may each be fed by adifferent reagent inlet.

Wherein the unit cell includes a cell capture site (e.g., in place of acolumn) as described further herein.

In certain aspects, the array downstream of a unit cell may be a digitalarray, i.e., an array that provides serial dilution to allowquantitation of a single target by digital PCR.

In certain aspects, an integrated microfluidic device may include: anarray of reaction sites; and a plurality of sample processing unit cellscomprising a plurality of sample processing sites, wherein the unit cellis in fluidic communication with a plurality of different reagentinlets; wherein sample inlets to the array are downstream of theplurality of sample processing sites of the plurality of unit cells.Such a microfluidic device is depicted in FIG. 17 and may or may notinclude an array of reaction sites downstream of the unit cells.

FIG. 17 is a schematic similar to that of FIG. 3 and showing anexemplary unit cell with a plurality of columns 1710 for retainingbeads, specifically a cleanup column 1710 a for depleting undesiredcomponents of a sample, and a plurality of capture columns 1710 b forcapturing different target molecules. Beads and sample may be flowedinto the cleanup column 1710 a through a first inlet 1704. Differentcapture beads may be loaded into different capture columns 1710 bthrough one or more bead inlets 1708.

In certain aspects, assay inlets may provide primers to amplify splintligation products formed as described herein. Alternatively or inaddition, assay inlets may provide sample barcode primers that bindsample barcode sequences (or their reverse complement) on ligationproducts from a specific sample in a pool of samples. Such samplebarcodes may be incorporated by splint ligation probes, and samples maybe pooled before being provided to the array through a sample inlet. Forexample, if 8 samples are pooled for each of 48 different sample inlets,and 8 different assay inlets each amplify ligation product from adifferent sample, then 48×8 (i.e., 386) different samples may be assayedin the array. As such, assay inlets may increase the number of targetsand/or samples detected.

For example, a method may include loading beads into a column of a unitcell and capturing sample (i.e., biomolecules of a sample such asproteins, antibodies, RNA, viral particles, etc.) on the bead (e.g.,before or after loading the beads into the column). As discussed herein,the bead may include (e.g., present on its surface) one or more of aprotein (e.g., an antibody, such as an antibody to a target serumprotein or viral antigen) and oligonucleotide (e.g., that hybridizes totarget RNA, such as a viral RNA). Additional steps may include washingbeads, such that a wash buffer flows over the beads in the column andinto a waste outlet. Optionally a reporter, such as anoligonucleotide-conjugated antibody that binds to target biomolecules oran oligonucleotide probe that hybridizes to target biomolecules may beflowed over the beads. Additional steps may include eluting from thebeads, such as by flowing an elution buffer over the beads in the columnand optionally further cycling the elution buffer across the beads suchas by passing the buffer around a loop using a peristaltic pump. Ofnote, mixing across sample processing sites in any step may be driven bya peristaltic pump. In certain aspects, elution may include degradingthe attachment of a biomolecule to the bead, such as by a restrictionenzyme, RNAse, or by a uracil DNA glycosylase (UDG) as described furtherherein. Eluted biomolecules may be mixed with a preamplification mastermix (e.g., that provides for whole genome amplification, wholetranscriptome amplification, or multiplexed targeted preamplification).Prior to, or in the same step as preamplification, a sample prep stepsuch as reverse transcription, proximity assay (such as proximityextension or ligation), or preparation of genomic DNA. In certainaspects, one or more enzymes (e.g., proteases) may be inhibited ordegraded prior to the preamplification or a later detection step. Incertain aspects, preamplification is not performed. Processed sample maythen be passed into an array of reactions sites of the same microfluidicdevice, for example, though a sample inlet from the unit cell into aplurality of sample chambers of different reaction sites as shown inFIG. 12. Different targets of a sample may be detected across differentreaction sites, for example, by PCR (e.g., qPCR) of products producedduring sample preparation.

The array IFC may have multiple layers such that sample and assay flowchannels can pass over one another. The array IFC may be elastomeric(e.g., include an elastomer such as PDMS), and may further haveelastomeric valves controlled through pressure applied to controlchannels (not shown) to deflect a membrane into a flow channel. Valvesmay be positioned along the dashed flow channels so as to contain sampleand/or assay in their respective chambers (e.g., preventing backwardscontamination after mixing). Valves may be positioned between pairs ofsample an assay chambers to control mixing (e.g., by interface freediffusion). Detailed descriptions of suitable array architecture may befound in US patent publications US20100120038 and US20140193896, both ofwhich are incorporated herein by reference in their entirety.

In certain aspects, such as for splinted ligation applications, asubject microfluidic workflow may include on or more of the followingsteps.

-   -   1. Load beads into the column of a unit cell from a shared inlet    -   2. Capture sample (e.g., from independent inlets)    -   3. Wash beads (e.g., from shared inlet)    -   4. Elute into a first chamber (e.g., from a shared inlet)    -   5. Load Preamp Mastermix into a second chamber (e.g., from        shared inlet)    -   6. Load Amplicon from preamp into sample chambers    -   7. Load assay mix (PCR mix, primers, and/or probes) into assay        chambers    -   8. Mix a fraction (i.e., at least a fraction of the contents of)        of sample chamber with assay mix in assay chamber

For example, a method of performing an assay on a microfluidic devicemay include each of:

loading beads into a column of a unit cell from a shared inlet;

capturing sample biomolecules of interest on the beads;

washing the beads;

eluting captured biomolecules into a first chamber;

loading preamp mastermix into a second chamber;

performing a preamplification reaction;

loading amplicon from preamplification reaction into sample chambers;

loading an assay mix into assay chambers; and

mixing at least a fraction of the contents of the sample chamber andassay chamber.

The microfluidic device may be an integrated microfluidic anmicrofluidic device as described above.

The step of capturing (capturing sample biomolecules on the beads) maybe before or after the step of loading the beads onto the column.Washing the beads may include flowing a wash buffer solution over thebeads in the column, or may include mixing the beads with a wash buffersolution and separating the beads from the solution (e.g., wherein thebeads are magnetic beads) before loading the beads on the column. Incertain aspects, beads that bind to different target biomolecules of asample may be combined before loading into the column, allowing formultiplexed sample processing and optionally downstream detection ofdifferent targets in each sample in an array of the microfluidic device.

As such, the subject methods and microfluidic devices may enabletargeted sample enrichment by: solid-phase bead-based capture ofpredefined nucleic acid sequences or other targets in columns of unitcells; integrated washing, elution, and preamp in sample processingsites of unit cells; and detection of specific targets (e.g. by qPCR)across a plurality of reaction sites for each sample.

Capture and/or Detection of Target Nucleotide Sequences

The assay mix may include at least one of PCR mix, primers, and a probe.The preamp master mix may include reverse transcriptase and apolymerase, such as when the target is an RNA such as a viral RNA.Reverse transcription and preamplification may be performed in the samestep.

The preamp mastermix may include primer pairs to a plurality ofdifferent target nucleotide sequences. The presence of each targetnucleotide sequence may be detected by PCR (such as by qPCR) after thestep of mixing. The plurality of different target nucleotide sequencesmay be viral RNA sequences.

The method may further include detecting the presence of thebiomolecules of interest after the step of mixing, such as by PCR (e.g.,end point or qPCR).

Alternatively or in addition, detection may be by sequencing. Forexample, the method may further include amplifying after the step ofmixing, and pooling the amplified product from different samples priorto sequencing. Preamplified sample may quantified by qPCR and normalizedfor pooling prior to the step of sequencing. The method may furtherinclude a bead cleanup step before and after preamplification, e.g.,using the same or different column of the unit cell. The method mayfurther comprising sample indexing after the step of mixing and beforepooling. FIG. 15 is a schematic showing exemplary loading schemes forRNA sequencing preparation (A) and DNA sequencing preparation (B).

The beads may specifically bind target viral particles (e.g., viralantigen), viral RNA, mammalian mRNA, genomic DNA, protein (e.g.,antibodies), or any other target biomolecule of interest. For example,herein the beads include and affinity reagent such as an antibody (e.g.,presented on the surface of the bead) that binds to a viral antigen ormammalian protein, such as a prostate specific antigen or other cancerbiomarker. Other suitable affinity reagents include an avidin or biotin,an aptamer, a tetramer such as an MHC or peptide-MHC, a receptor such asa TCR, and so forth. In certain aspects the beads may include a nucleicacid, such as an ssDNA that specifically binds a target nucleotidesequence as described further herein.

FIG. 16A provides a schematic showing exemplary loading scheme foroligonucleotide detection on chip (such as for detection of a viralRNA). Of note, an array may be downstream of the sample processing unitcell such that one or more target viral RNA sequences can be detected byqPCR as described herein.

In certain aspects, the biomolecules of interest include one or moretarget nucleotide sequences. For example, the bead is functionalizedwith single stranded DNA sequences that specifically hybridize the oneor more target nucleotide sequences. The one or more target nucleotidesequences may be a viral polynucleotide sequence, an RNA sequence, or aviral RNA sequence. For example, the viral RNA sequence may be anSARS-CoV-2 viral RNA sequence, e.g., wherein the one or more targetnucleotide sequences include at least two of N1, N2, and N3 SARS-CoV-2sequences, and the method may include detecting the at least two of N1,N2, and N3 SARS-CoV-2 sequences in separate reaction sites.Alternatively or in addition, the viral RNA sequence may be an influenzaRNA sequence, e.g., wherein the one or more target nucleotide sequencesinclude at least an H3N2 Influenza RNA sequence and an H1N1 InfluenzaRNA sequence in separate reaction sites, and the method may includedetecting at least the H3N2 Influenza RNA sequence and the H1N1Influenza RNA sequence in separate reaction sites. As discussedelsewhere herein, the reaction site includes a sample chamber and anassay chamber.

In certain aspects, a unit cell may include multiple columns each loadedwith beads that capture a different biomolecule of interest (e.g., adifferent target protein or nucleotide sequence).

Capture and/or Detection of Sample Proteins

Sample proteins, such as cancer markers or antibodies to a pathogen, maybe captured, processed, and/or detected as described further herein.

FIG. 16B provides a schematic showing exemplary loading scheme forsample preparation for detection of a protein (such as a cancer marker,viral antigen, or antibody to a viral antigen). Of note, an array may bedownstream of the sample processing unit cell such that one or moretarget proteins can be detected by (e.g., by immune-qPCR as describedherein, in which case harvest solution may not be needed). In caseswhere sequencing is not the method of detection, tagmentation buffer maynot be needed.

In certain aspects where the beads include an antibody that binds to aviral particle, The method may further include detecting viral RNA asdescribed further herein, or the method may further include detectingthe viral particle (e.g., by an immuno-PCR as described further herein).For example, oligonucleotide conjugated antibody may be bound to theviral particle, and the oligonucleotide amplified by PCR (e.g., detectedby qPCR).

Microfluidic detection of rare species can often require expensive,contamination-prone sample preparation in order to provide competitiveassay sensitivity compared to similar tube- or microwell plate-basedassays. As an alternative to these sample preparation steps, solid-phasesample enrichment integrated within the microfluidic device has provensufficient for some workflows (e.g. for mRNA and bacterial genomesequencing). Using similar methods, solid phase capture can be used toenrich for the presence of viral particles. As described herein, viralparticles can be captured using beads (the solid phase) that have beenconjugated with biomolecules, which specifically target correspondingbiomolecules of the viral particles of interest. For example,antigen-antibody or receptor-ligand interactions. The beads can then beused to concentrate the population of viral content into a very smallvolume that can be used for nano- to microliter scale automateddetection by qPCR all within a single device that completes thesample-to-answer (e.g., in less than 3 hours).

In certain aspects where the beads include a viral antigen (e.g., wholevirus or portion thereof, such as SARS-CoV-2 spike S1 and/or S2), thepresence sample antibodies that bind to the viral antigen may bedetected. For example, antibodies from serum, plasma, whole blood,saliva, or a nasal swab may be passed over such beads in a column. Anoligonucleotide conjugated antibody may then be bound to the sampleantibodies, and the oligonucleotide may then be detected (e.g., by qPCRsuch as in an immuno-PCR workflow, or by sequencing). In certainaspects, the method may include detecting different antibody types, suchas IgG and IgM (e.g., by immuno-PCR of a secondary antibodies thatspecifically bind an antibody type). In certain aspects, separatecolumns of a unit cell each include beads presenting a different viralantigen, e.g., wherein each different viral antigen is from a differentstrain (e.g., a different mutation of the SARS-CoV-2 spike S1 and/or S2protein domains). The method may further include using a first columnfor cleanup, such as a serum based cleanup to produce a purified serumsample (as described further herein). The method may further includesplitting the purified sample between separate columns that each includebeads presenting a different viral antigen. Such different antibodytypes and/or antibodies to different viral antigen may be detected inthe unit cell (e.g., using different color probes) or may be detected indifferent reaction sites downstream of the unit cell. Detection may beby PCR, such as qPCR, using primers specific for oligonucleotideconjugated to a specific secondary antibody.

Beads may be loaded into the column of a unit cell from an inlet sharedwith the sample (e.g., beads can be loaded before sample, or they can beloaded in admixture such as with sample biomolecules already captured onthe beads)., The sample biomolecules of interest may include proteins.The biomolecules of interest may be captured by flowing sample over thebeads loaded into the column, or the biomolecules of interest may becaptured by mixing the beads with a sample before loading the beads intothe column. The sample proteins may be captured on the beads byantibodies bound to the beads. The method may further include bindingantibodies to the sample proteins captured on the beads, wherein theantibodies are conjugated to oligonucleotides. In certain aspects,sample may be blood (e.g., serum, plasma, or whole blood), saliva or anasal swab. When the sample is serum, the method may further include aserum cleanup step in a first column of the unit cell. If the sampleproteins are antibodies, the beads may present an antigen such as aviral antigen. For example, the viral antigen may be a SARS-CoV-2antigen such as a SARS-CoV-2 spike S1 and/or S2 protein domain orpeptide thereof. In another example, the viral antigen is an influenzaantigen. In certain aspects a unit cell includes a plurality of columnsthat are each loaded with beads presenting a different viral antigen.For example, the different viral antigens include variants of the samevirus, such as different SARS-CoV-2 spike S1 and/or S2 domain mutants orpeptides thereof. The method may further include detecting theantibodies by immuno-PCR. The method may include separately detecting atleast IgG and IgM antibodies specific for a viral antigen.

The method may include detecting the presence of the proteins in aplurality of reaction sites of the microfluidic device, such as byimmuno-PCR or a proximity assay. For example, the method may includehybridizing an ssDNA complement to the oligonucleotide of anoligonucleotide conjugated antibody, and may further include cleaving(degrading) the oligonucleotide. For example, the oligonucleotidesconjugated to the antibodies include uracil, and wherein degrading iswith Uracil DNA-glycosylase (UDG).

In certain aspects a plurality of different proteins are detected foreach of a plurality of different samples. A step of detecting mayinclude PCR, such as qPCR, in the unit cell, or in an array of reactionsites downstream of the unit cell. In certain aspects, the sampleproteins are cancer biomarkers such as prostate specific antigens (e.g.,PSA, free PSA p2PSA, and/or other isoforms of PSA) and the beads includeantibodies to the cancer biomarkers. In other aspects, the sampleproteins are antibodies (e.g., to a viral antigen) and the beads includethe antigen specifically bound by the antibodies.

Methods may further include performing a bead based cleanup, such as aserum cleanup to produce a purified serum sample off the microfluidicdevice. As such, the unit cell includes at least two columns, whereinone of the at least two columns is used for the cleanup prior to furthersample processing. In certain aspects, a serum based cleanup depletes atleast one of IgG and albumin by binding to beads. For example, beadswith antibody specific for human serum albumin and/or protein G forcapturing IgG, such as PureProteome beads, may be used.

In certain aspects, separate columns of a unit cell are used to enrichfor a separate biomolecule of interest from the same sample. Forexample, a first column may be used for cleanup (e.g., serum basedcleanup to produce a purified serum sample), and a method may furtherinclude splitting the purified sample between separate columns that eachenrich a different biomolecule of interest, which may then be detectedthrough PCR (e.g., qPCR) in sample processing sites or reaction sitesdownstream of the column. Alternatively, processed sample may beharvested and run on a separate microfluidic device comprising an arrayof reaction sites. The workflow may be performed for immuno-PCR,proximity assays, or reverse transcribed RNA targets.

A serological method of the subject application may include detectingantibodies to a pathogen, such as a to a virus. In one example, theantibodies may be to SARS-CoV2, such as an S1 or S2 domain of theSARS-CoV-2 spike protein. As such, a sample for the subject methods maybe prepared by:

-   -   Block 100-200K SARS-CoV2 antigen-beads per sample with Blocking        buffer (1% BSA, PBS)    -   Incubate for 1 h at RT with agitation (1500 rpm).    -   Wash with Washing Solution (1× PBS, 0.1% BSA, 0.01% Tween 20)    -   Use a tube/plate magnetic separator or by centrifugation to        separate the beads from the solutions    -   Dilute each bead pellet to reach a concentration of 2-4×10⁶        Bead/mL    -   Aliquot the beads in each tube or well    -   Incubate with Target Antibody (for spiked-in samples) OR with        TEST SAMPLE    -   Add Target Antibody dilutions (anti-RBD from Bethyl or        anti-Spike S1 from Sino) OR the TEST SAMPLES    -   Incubate 2 hours at RT @1500 rpm    -   Wash with Washing Solution, separate the beads from the solution        with Tube/Plate Magnetic Separator or by centrifugation    -   Beads may then be loaded and sample process in a microfluidic        device as described herein

As described elsewhere herein, FIG. 17 is a schematic similar to that ofFIG. 3 and showing an exemplary unit cell with a plurality of columns1710 for retaining beads, specifically a cleanup column 1710 a fordepleting undesired components of a sample, and a plurality of capturecolumns 1710 b for capturing different target molecules. Beads andsample may be flowed into the cleanup column 1710 a through a firstinlet 1704. Different capture beads may be loaded into different capturecolumns 1710 b through one or more bead inlets 1708.

FIG. 18 shows an exemplary cleanup step which may be performed in thecleanup column 1710 a of FIG. 17. Specifically, FIG. 18 shows depletionof IgG and Albumin from serum.

FIG. 19 shows an exemplary capture, sample preparation and PCRamplification performed in one of the capture columns 1710 b and seriesof processing sites 1714 of FIG. 17. Specifically, FIG. 19 shows captureof a target sample protein by beads presenting an antibody to the targetprotein, washing of the beads, binding and washing of an oligonucleotideconjugated antibody to the protein captured by the beads. FIG. 19further shows annealing of a complementary oligonucleotide, elution ofthe oligonucleotides (through cleavage by UDG of one or more uracils ofthe oligonucleotide conjugated to the antibody). PCR of the elutedoligonucleotides can allow for direct detection such as by qPCR asdescribed herein, or amplified product can be sequenced (e.g., sampleindexed in the microfluidic device, pooled and sequenced). In general,immuno-PCR or a proximity assay may be used to detect target sampleproteins.

A biomarker detection method of the subject application may, forexample, include automated detection of specific biomarkers such as PSAs(prostate specific antigens, e.g., total PSA, free PSA and pro-2-PSA).These biomarkers are used in an FDA approved index, the Prostate HealthIndex (PHI) formula, to measure the risk of/potentially identifyprostate cancer from serum samples. immunoqPCR can be adapted to providequantitative output for these specific biomarkers in the microfluidicworkflows described herein, using antibodies that specifically detecteach of these target biomarkers, coupled with antibody-DNA tags; DNAtags can be separated out and quantified using PCR. For example, such aworkflow may use the microfluidic device shown in FIG. 3 or FIG. 17,optionally integrated with the array such as that shown in FIG. 12. Asuch, a serum sample input may be followed by on-chip serum clean up,target capture and PCR. On-chip serum clean up may reduce the need forsample handling prior to loading the IFC, as sample clean up is carriedout in an automated manner, on-chip. The cleaned serum sample may besplit to detect the presence of the 3 PSA biomarkers. One sample inputper unit, on the IFC, provides 3 different PSA outputs for each specificPSA PHI biomarker, in an automated manner. A set of PCR dilution outputscan be detected for each of these 3 different PSA outputs, individuallyand quantitatively, to be used in the PHI calculation (e.g., thecalculation may be presented to a user by software running themicrofluidic workflow and collecting the PCR dilution outputs).

As described further herein, an antibody sandwich assay type format mayinclude a target capture antibody (e.g., on beads of the subjectapplication) and a universal secondary antibody (e.g., that binds thePSA). In immune-PCR, antibody may be conjugated to either a single ordouble stranded DNA, and PCR can be performed whilst the DNA is stillattached to the antibody or after it is cleaved off. For example, targetcapture antibody may be biotinylated and bound to a streptavidin bead,sample is flowed over the bead and PSA biomarker(s) are bound to thebead, secondary antibody tagged with a single strand oligonucleotidesequence is bound to the PSA biomarker(s), a complement to the singlestrand oligonucleotide sequence is bound to the tag on the secondaryantibody to produce a double stranded DNA tag, the double strand DNA tagis separated from the antibody, and a PCR (e.g., qPCR) on the doublestranded DNA is performed.

As such, a subject method may include one or more of a modifiedimmunoqPCR for detection and quantitation, conjugated Bead-Ab capture ofPHI target protein panel, a single microfluidic device for an integratedworkflow, a multiplexed bead capture setup, a medium throughput witheach sample input produces an automated multi-biomarker panel output,and/or serial stages of bead capture on the microfluidic device. Whilethe above example is for PSA biomarkers, is would be understood than anysuitable biomarkers may be analyzed by this method.

Bead Based Purification

One or more rounds of bead cleanup (bead based purification of samplebiomolecules) may be performed in any of the methods described herein.Bead based purification (or “cleanup”) generally refers to theenrichment or removal of a class of biomolecules, such a common protein(e.g., IgG, albumin, etc.) or an oligonucleotide (e.g. RNA and/or DNA).Aspects include at least two rounds of bead-based purification (e.g., ofoligonucleotides). For example, a subject method may include performinga round of bead-based purification of oligonucleotides (e.g., sampleoligonucleotides) before an amplification reaction, and another round ofbead-based purification (e.g., of amplified product) after theamplification reaction. The amplification reaction may be any reactiondescribed herein, for example, may be a preamplification reaction forsequencing preparation.

A round of bead-based purification may include capturing polynucleotideson beads, washing of the beads with a capture buffer, washing the beadswith an alcohol, and allowing the alcohol to evaporate through one ormore PDMS layers of the elastomeric device. In certain aspects, thealcohol is ethanol. In certain aspects, the beads extract RNA, DNA orboth.

FIG. 13 is an image of an exemplary elastomeric microfluidic device andexemplary loading scheme of the subject application, similar in somerespects to that of FIG. 2, but overlaid with markings showing wasteoutlets 1304, sample and bead inlets 1306, elution buffer inlets 1308,ethanol inlets 1310, PCR mix inlet 1312, harvest outlets 1312, harvestbuffer inlets 1314, and capture buffer (also used as wash buffer) inlets1316. Such a loading scheme may be used for bead cleanup, such as formultiple rounds of bead cleanup (such as before and after anamplification reaction).

FIG. 14 is schematic similar to that of FIG. 3 and showing directions offlow from inlets, outlets and within the unit cell such as in a loadingscheme of FIG. 13. Individual steps are described further herein.

A bead cleanup method may include on or more of the following steps:

-   -   1. Capture beads that have been pre-loaded with sample (e.g., by        flowing sample through the column and flowing unbound sample to        a waste outlet).    -   2. Wash beads with capture buffer.    -   3. Wash beads with ethanol.    -   4. Dry beads (e.g., under heat).    -   5. Elute into a processing site (e.g., by flowing elution buffer        from an inlet through the capture site and into a first sample        processing site).    -   6. Optionally resuspend sample and beads into sample inlet using        capture buffer and repeat steps 1 through 5 in another round of        cleanup.    -   7. Add amplification mix into a second sample processing site,        and mix with the sample in the first sample processing site.    -   8. Amplify the sample (e.g., PCR amplification).    -   9. Resuspend sample and beads into sample inlet using capture        buffer.    -   10. Repeat steps 1 through 5 in a post-amplification bead        cleanup.    -   11. Optionally resuspend sample and beads into sample inlet        using capture buffer and repeat steps 1 through 5 in another        round of cleanup.    -   12. Flow harvest buffer through the column (e.g., through the        entire unit cell) and into a harvest outlet.

The harvested amplified sample may be at a purity sufficient for librarypreparation and sequencing. In certain aspects, the amplification stepincludes sample indexing and/or qPCR for normalization prior to poolingharvested samples.

Cell Capture, Processing and Detection

In certain aspects, an array IFC may be integrated with a unit cellcomprising a plurality of sample processing sites (e.g., chambers and/orloops), wherein the unit cell further includes a cell capture site(e.g., in place of a column in any such embodiments described herein).The cell capture site may include one or more bypass channels. Forexample, the unit cell may include architecture and be used as describedin US patent publication 20130296196, which is incorporated herein byreference. Some single cell processing may include specific targetamplification, whole genome amplification, whole transcriptomeamplification, real-time PCR preparation, copy number variation,preamplification, and/or mRNA sequencing preparation. In certainaspects, a single cell may be captured (isolated), lysed, and thenprotein and/or RNA of the cell may be detected, for example as describedin US patent publication number US20150132743, which is incorporatedherein by reference. In the context of the subject application, a cellcaptured in a unit cell comprising a cell capture site may then belysed, and optionally subjected to one or more additional reactions suchas reverse transcription, proximity assay for detecting protein targets(e.g., proximity extension or ligations), and/or preamplification (e.g.,whole genome amplification, targeted multiplexed preamplification ofgDNA, cDNA or proximity extension products, etc.) prior to flowing theprocessed sample into an array of reaction sites of the samemicrofluidic device, where different RNA, DNA and/or protein targets mayeach be detected in separate reaction sites.

The cell capture site may selectively capture cells based on size. Forexample, cells 5 microns or less in diameter are captured with less than5% (e.g., less than 1%) of the efficiency that cells larger than 10microns are captured. The cell capture site may selectively capturecells based on affinity binding, such as binding of an antibodyimmobilized in the capture site to a cell expressing the correspondingantigen on its surface (e.g., so as to enrich for a particular celltype, such as an immune cell type such as T-cells or B-cells or subsetsthereof). Individual unit cells may be in fluidic communication withsample inlet channels of the array architecture comprising a pluralityof reaction sites, such as is shown in FIG. 12. In certain aspects, thecaptured cell may be lysed in a first step. Cell lysis may be followedby any of the reactions described herein in the context of a unit celland/or an array of reaction sites. For example, after lysis, RNA fromthe cell may be reverse transcribed and preamplified, or genomic DNA maybe processed and preamplified. Preamplification may be may be targeted,such as through a multiplexed reaction with at least 4 different primerpairs that each amplify a different target nucleotide sequences. Theprocessed cell lysate (e.g., preamplified cell lysate) may be flowedinto a sample inlet of an array of reaction sites, and a differenttarget nucleotide sequence may be detected in different reaction sitesusing different primer pairs and/or different target specific probes.

For example, a single cell workflow may include flowing the plurality ofcells through the microfluidic device such that individual cells fromthe plurality of cells are capture at individual capture sites indifferent unit cells of the microfluidic device; lysing the plurality ofcaptured individual cells at the individual capture sites of themicrofluidic device; performing reverse transcription, within themicrofluidic device, on the plurality of individual lysed cells toproduce reverse transcription products associated with each respectiveindividual cell; optionally performing a multiplexed preamplification ofcDNA produced by reverse transcriptions; splitting the contents of aunit cell across multiple reactions sites in an array of themicrofluidic device; performing PCR, such as qPCR, within themicrofluidic device to detect different targets (e.g., different reversetranscription products) in different reaction sites.

Alternatively or in addition, a single cell workflow may include flowingthe plurality of cells through the microfluidic device such thatindividual cells from the plurality of cells are capture at individualcapture sites in different unit cells of the microfluidic device; lysingthe plurality of captured individual cells at the individual capturesites of the microfluidic device; incubating the cell lysate with two ormore proximity extension probes in a binding reaction at an incubationtemperature from about 15° C. to about 50° C. for a length of time fromabout 5 minutes to about 6 hours under conditions where the proximityextension probes bind to the target analyte, if present, in the celllysate; incubating the binding reaction with an extension mix thatincludes a polymerase, wherein hybridized oligonucleotide components ofthe proximity extension probe are extended by the polymerase to produceextension products; splitting the contents of a unit cell acrossmultiple reactions sites in an array of the microfluidic device;performing PCR, such as qPCR, within the microfluidic device to detectdifferent targets (e.g., different proximity extension products) indifferent reaction sites.

Kits for an Integrated Workflow

The subject application also includes kits for performing any of theabove methods. For example, a kit may include a microfluidic device ofany one of embodiments and/or may include one or more reagents forperforming the methods of any one of the above methods. Such reagentsmay be selected from one or more of an RNAse inhibitor, reversetranscriptase, polymerase, a preamplification mix (e.g., comprising aplurality of primer pairs that specifically amplify different targetnucleotide sequences), beads that specifically capture samplebiomolecules of interest as described herein, primers, probes,oligonucleotide-conjugated antibodies, enzymes of cleavingoligonucleotide from the antibodies they are conjugated to, or any othersuitable reagent for performing the methods of the subject application.

Sample Barcoding Methods

Sample barcoding (i.e., sample tagging, or encoding) may increase samplethroughput but the leftover primers (e.g., from sample with little or notarget) may create crosstalk, leading to a false positive and/or higherbackground. Discussed herein are methods and kits for reducing suchcrosstalk.

FIG. 20 shows a multiplex sample barcoding workflow of the subjectapplication. Specifically, target nucleotide sequences from differentsamples can be reverse transcribed (if RNA) and preamplified in areaction that incorporates a sample tag (i.e., sample barcode sequence).Preamplified mixture from different samples can be pooled and loadedonto a single inlet of a microfluidic device, then split into differentchambers (different reaction sites) where target nucleotide sequenceswith different sample tags are selectively amplified. Such a workflowincreased the number of samples that can be loaded onto a microfluidicdevice for a given number of sample inlets. The specific detection ofeach sample tagged target nucleotide sequence prevents the need toretest all samples individually if at least one is positive for a targetnucleotide sequence. Each reaction site has the mixture of preamplifiedsamples, a target specific probe (e.g., that fluoresces upon binding tothe target), a sample barcode primer that selectively amplifies thereaction product of a specific sample, and a reverse primer (e.g., thatis target specific, or that is specific for a target barcodeincorporated during the preamplification reaction, such if when multipletargets are detected for a sample).

FIG. 21 shows a simple Dorfman pooling method in which samples are notbarcoded and are not split into separate reaction sites after mixing.Retesting individual samples if the pool of samples is positive for atarget nucleotide sequence requires additional steps and reagents.

FIG. 22 shows the efficiency of the multiplex sample barcoding (mpe) andDorfman pooling (pe) methods when 4 samples are mixed (A) or 8 samplesare mixed (B). An increase in efficiency can allow for a reduction insample handling, in reagents, and in space used on the microfluidicdevice.

FIG. 23 shows the mechanism by which crosstalk can occur when using themultiplexed sample barcoding approach of FIG. 20. Specifically, leftovertagged target specific primers may react with tagged target nucleotidesequences from another sample after the samples are mixed, leading tobackground.

FIG. 24 provides a reaction scheme in which leftover primer from anegative sample (i.e., sample B that does not have a target nucleotidesequence) may react with the preamplified target nucleotide sequencefrom a positive sample (sample A) after the samples are mixed together,leading to background (e.g., leading to a lower cycle threshold (CT) ina reaction site for detecting the target in sample B). The probe doesnot compete with the leftover primer.

FIG. 25 provides a reaction scheme in which target specific probecompetes with leftover primer (i.e., with barcoded target specificprimer, also referred to herein as tagged target specific primer) forbinding to the preamplified target nucleotide sequence reducescrosstalk.

FIG. 26 shows qPCR curves for a set of four samples in triplicate inwhich the first sample (black line) is positive and the other samplesare negative for a target nucleotide sequence.

FIG. 26 shows qPCR curves for a set of four samples in triplicate inwhich the first sample (black line) is positive and the other samplesare negative for a target nucleotide sequence, under the scheme of FIG.24.

FIG. 27 shows qPCR curves for a set of four samples in triplicate inwhich the first sample (black line) is positive and the other samplesare negative for a target nucleotide sequence, under the scheme of FIG.25, demonstrating a CT increase of 2 for negative samples compared toFIG. 26.

In certain aspects, an assay method for detecting at least one targetnucleic acid in a plurality of samples includes:

-   -   a) reverse transcribing and preamplifying a target nucleotide        sequence in each of S separate samples to produce a tagged        target nucleotide sequence from each sample,        -   wherein at least one of the S samples includes the target            nucleotide sequence,        -   wherein the tagged target nucleotide sequence includes a            sample tag and a target nucleotide sequence,        -   wherein preamplifying is with a tagged target-specific            primer that includes a sample tag and a target-specific            sequence, and        -   wherein the target-specific sequence hybridizes to a portion            of the target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) splitting the mixture into a plurality of reaction sites;    -   d) adding different primer pairs to each reaction sites;    -   e) amplifying the tagged target nucleotide sequence from a        different sample in each reaction site,        -   wherein each different primer pair includes a primer that            hybridizes to a different sample tag; and/or    -   f) detecting the presence of the of the amplified tagged target        nucleic acid by qPCR with a fluorescent target-specific probe        that includes at least a portion of the target-specific sequence        but does not include a sample tag;        -   wherein step e of amplifying is in the presence of the            target-specific probe.

More generally, an assay method for detecting at least one targetnucleic acid in a plurality of samples may include:

-   -   a) separately subjecting each of S samples to an encoding        reaction that produces a tagged target nucleotide sequence using        at least one tagged target-specific primer, wherein at least one        of the S samples includes the target nucleotide sequence (i.e.,        that hybridizes to a strand of the target nucleotide sequence)        and wherein the tagged target nucleotide sequence includes a        sample tag and a target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) splitting the mixture into a plurality of reaction sites;    -   d) adding different primer pairs to different reaction sites,        wherein each different primer pair includes a primer that        hybridizes to a different sample tag to amplify a tagged target        nucleotide sequence from a specific sample;    -   e) amplifying the tagged target nucleotide sequence from the at        least one of the S samples in the presence of a target-specific        probe, wherein the target-specific probe includes a sequence        identical to at least a portion of a target-specific sequence of        the target-specific primer but does not include a sample tag;        and/or    -   f) detecting the presence of the tagged target nucleotide.

The target-specific probe may include sequence identical to at least 6nucleotides (e.g., at least 12 nucleotides long, or at least 18nucleotides long, such as between 6 and 30 nucleotides long) of thetarget-specific sequence of the target-specific primer. Alternatively orin addition, the tagged target nucleotide sequence may include a sampletag that is at least 4 nucleotides long, (e.g., at least 6 nucleotideslong, at least 12 nucleotides, or at least 18 nucleotides long, such asbetween 6 and 30 nucleotides long). Alternatively or in addition, thetarget-specific sequence is at least 6 nucleotides long (e.g., at least12 nucleotides long, or at least 18 nucleotides long, such as between 6and 30 nucleotides long). Alternatively or in addition, the targetnucleotide sequence may be at least 50 nucleotides long (e.g., at least100 nucleotides long, at least 150 nucleotides long, such as at orbetween 5o and 300 nucleotides long).

In certain aspects, step a) includes reaction with a tagged primer thatincludes the sample tag but does not include the target nucleotidesequence, for example, wherein the tagged primer is at a higherconcentration than the tagged target-specific primer.

A target-specific primer that does not include the tag and is thereverse complement to a portion of the target nucleotide sequence may beused (e.g., to reverse transcribe and/or amplify the tagged targetnucleotide sequence). Step a) may further includes reverse transcribingthe target nucleotide sequence using the target-specific primer.

In certain aspects the the tagged target-specific primer includesuracil, such as when the method further includes adding a uracilN-glycosylase (UDG) to the mixture of tagged target nucleotide sequencesto degrade leftover tagged target specific primer.

In certain aspects, at least one of the S samples includes the targetnucleotide sequence and at least one of the S samples does not includethe target nucleotide sequence

A method may further include reverse transcribing the tagged targetnucleotide sequence, using a target-specific primer, prior to step a).Alternatively, step a) may include preamplifying the tagged targetnucleotide sequence (e.g., in the same reaction).

In certain aspects, step e) may be performed at least in duplicate. Incertain aspects, at least one of the S samples does not include thetarget nucleotide sequence. Step f) of detection may be by PCR, such asendpoint PCR or qPCR. When detection is by qPCR, the target-specificprobe (e.g., its completion with the tagged target specific primer) mayincrease the CT by at least 1, at least 2, at least 4, or at least 6 ina reaction site where the primer pair is specific for a sample that doesnot include the target nucleotide sequence. For example, the presence ofthe target-specific probe increases the dCT between a reaction sitewhere the primer pair is specific for a sample that does not include thetarget nucleotide sequence and a different reaction site where theprimer pair is specific for a sample that does not include the targetnucleotide sequence by at least a dCT of 1, 2, 4 or 6, such as a 20%increase in dCT or a 40% increase in the dCT. In certain aspects, thetarget specific probe may reduce binding of leftover tagged targetspecific primer to tagged target nucleotide sequence by at least 25%, atleast 50%, or at least 75%. In certain aspects the probe may be in atleast 5 times, at least 10 times, or at least 20 times in excess ofleftover tagged target specific probe in the mixture.

In certain aspects, step f) of detecting includes detecting a signalfrom the target-specific probe. For example, the probe may include afluorophore and optionally further a quencher, e.g., such that thefluorophore is quenched when the probe is not hybridized to the targetnucleotide sequence and such that the probe fluoresces uponhybridization.

Any of the method steps may be performed on a microfluidic device of thesubject application. In certain aspects, least steps e) and f) areperformed on an array microfluidic device including an array of reactionsites. For example, at least steps c) through f) are performed on anarray microfluidic device including an array of reaction sites. Inanother example, all of steps steps a) through f) are performed on anintegrated microfluidic device including sample processing unit cellsand an array of reaction sites. Such an integrated microfluidic devicemay be of any embodiment described herein. For example, individualreaction sites of the array of reaction sites of the device may includea unique combination of a sample inlet and a reagent inlet. In certainaspects, step c) includes flowing the mixture of step b) into a sampleinlet of the array microfluidic device. In certain aspects, the numberof inlets to a microfluidic device may be restricted based on physicallimitations such as the substrate of the device or carrier, the pressureneeded to drive fluid through small channels, and/or alignment of wellsin a carrier of the device with inlet channels in the microfluidicdevice. As such, dense arrays (e.g., including more than 200 reactionsites across a square centimetre) may not have enough inlets to direct adifferent sample to each reaction site. As such, sample barcoding of thesubject application can provide the ability to pool sample barcodedsamples, flow them through the same channel, and detect the presence ofa target with a different sample barcode in different reaction sites.

In certain aspects, target-specific probe does not include a label(e.g., it is a competition probe that does not provide any fluorescentsignal). In such aspects, step f) of detecting is with a labelled targetspecific probe that does not compete with the tagged target-specificprimer for binding to the target specific nucleotide sequence. Forexample, step f) of detecting is with an intercalating dye such as SYBRGreen.

In certain aspects the number of samples S is at least 2, at least 4, atleast 8, or at least 16, such as at or between 4 and 8.

Method of sample barcoding may further include flowing the mixture oftagged target nucleotide sequences from step c through a single channelthat splits into the plurality of reaction sites of step d. For example,the single channel may be a sample inlet to a plurality of reactionsties, e.g., each reaction site including a sample chamber and a assaychamber as described herein. The plurality of reaction sites areseparate locations of an array microfluidic device, and the method mayfurther include fluidically isolating the reaction sites from oneanother prior to step e of detecting the tagged target nucleic acids.

In certain aspects T different target nucleotide sequences in the samesample are tagged with the same sample tag but are detected in separatereaction sties. The tagged target nucleotide sequence may include aunique combination of a sample tag and a target-specific tag. Forexample, step a) may further include a target specific reverse primerthat includes the target-specific tag but does not include the sampletag. A reaction site may amplify a specific target from a specificsample using a primer to the sample tag and a primer to thetarget-specific tag. A reaction site may amplify a specific target froma specific sample using a primer (e.g., reverse primer) to the sampletag and a primer to the target nucleotide sequence. Optionally further,each target is detected with a target-specific probe. Step e) ofamplifying may include loading each reaction site with a primer pairspecific for a particular combination of a sample tag and a targetspecific tag, e.g., such that wherein each of the S×T combinations areamplified in a separate reaction site. In certain aspects, T may be atleast 3, at least 4, or at least 6. In certain aspects, the targetnucleotide sequence is a viral nucleotide sequence, such as a viral RNAsequence (e.g., an influenza or SARS-CoV-2 viral RNA sequence). Forexample, the T different target nucleotide sequences include an H3N2Influenza RNA sequence and a H1N1 Influenza RNA sequence. Alternativelyor in addition the T different target nucleotide sequences include atleast two of an N1, N2, and N3 and SARS-CoV-2 sequence.

As described herein, the sample may be any biological sample such as ablood sample (e.g., serum, plasma or whole blood), saliva, a nasal swab,or derived from solid tissue.

Sample Barcoding for Sequencing and/or PCR Detection

Crosstalk may also occur when sample indexing (barcoding) prior tomixing and sequencing, in particular when there are amplification stepsafter mixing indexed samples. An assay method for detecting at least onetarget nucleic acid in a plurality of samples may include:

-   -   a) separately subjecting each of S samples to an encoding        reaction that produces a tagged target nucleotide sequence using        at least one tagged target-specific primer, wherein at least one        of the S samples includes the target nucleotide sequence and        wherein the tagged target nucleotide sequence includes a sample        tag and a target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) amplifying the tagged target nucleotide sequence from the at        least one of the S samples in the presence of a target-specific        probe, wherein the target-specific probe includes a sequence        identical to at least a portion of a target-specific sequence of        the target-specific primer but does not include a sample tag;        and/or    -   d) detecting the presence of the tagged target nucleotide.

In certain aspects step d) of detecting is by sequencing the amplifiedtagged target nucleotide sequences. Step a) of encoding may includeincorporating a sequencing adaptor sequence into the tagged targetnucleotide sequence. Step a) may be performed on a microfluidic deviceincluding sample processing unit cells, and tagged target nucleotidesequence may be collected from the microfluidic device before step b) ofmixing. Alternatively or in addition, step d) of detecting includes PCR(e.g., qPCR).

Kits for Sample Barcoding

The subject application also includes kits for performing any of theabove methods. For example, a kit may include a microfluidic device ofany one of embodiments and/or may include one or more reagents forperforming the methods of any one of the above methods. Such reagentsmay be selected from one or more of an RNAse inhibitor, reversetranscriptase, polymerase, a preamplification mix (e.g., including aplurality of primer pairs that specifically amplify different targetnucleotide sequences), beads that specifically capture samplebiomolecules of interest as described herein, primers, probes,oligonucleotide-conjugated antibodies, enzymes of cleavingoligonucleotide from the antibodies they are conjugated to, or any othersuitable reagent for performing the methods of the subject application.

A kit for detecting at least one target nucleic acid in a plurality ofsamples may include:

a tagged target-specific primer for each of S samples, wherein eachtagged target-specific primer includes a sample tag and atarget-specific sequence, and

a target-specific probe that includes at least a portion of thetarget-specific sequence;

wherein each of the tagged target-specific primers and the probe are inseparate partitions.

The kit may further include one or more of a strand displacingpolymerase (e.g., that displaces the probe when during an amplificationreaction), a reverse transcriptase, and RNAse inhibitor, or any buffers,master mixes or other components the for subject methods.

In certain aspects, the tagged target-specific primer is in mixture witha target specific reverse primer that and does not include a sample tag.The reverse primer may include a target specific tag. The reverse primerwould hybridize the revere complement of the target nucleotide sequencestrand that the tagged target-specific primer hybridizes to. In certainapplications, the reverse primer hybridizes to an mRNA target-specificsequence to enable reverse transcription.

The kit may further include a set of S different primers that eachhybridize to a different sample tag, wherein each of the S differentprimers is in a separate partition. At least some of the S differentprimers may be in admixture with a target specific reverse primer. Thekit may further include a target specific reverse primer, e.g., whereinthe target specific reverse primer does not include a sample tag.

The probe and/or primers of the kit may be of any of the aspectsdescribed for the methods herein. The kit may further include amicrofluidic device of any aspects of the subject application. Themicrofluidic device is and elastomeric device. The microfluidic devicemay be an array device including a plurality of reaction sites, e.g.,wherein individual reaction sites include a unique combination of asample inlet and a reagent inlet. In certain aspects the microfluidicdevice further includes sample processing unit cells, for example, whena plurality of samples bound to beads are mixed and an encoding reactionand/or preamplification of encoded products is performed on themicrofluidic device (e.g., by splint ligation workflow describedherein).

3 Primer Sample Barcoding

Another approach to reducing crosstalk in sample barcoding is a threeprimer approach described herein.

FIG. 28 shows another approach to reducing cross talk, in which UDG isused to degrade leftover primer (top) and the probe (GSP) does notcompete with leftover primer. Alternatively or in addition, theconcentration of the tagged target specific primer can be below theconcentration of a tag primer that is not target specific, such that thetagged primer takes. The tagged primer would not be expected to createcrosstalk after mixing, as it is not target specific.

In certain aspects, an assay method for detecting at least one targetnucleic acid in a plurality of samples may include:

-   -   a) separately subjecting each of S samples to an encoding        reaction that produces a tagged target nucleotide sequence using        at least one tagged target-specific primer, wherein at least one        of the S samples includes the target nucleotide sequence;    -   b) mixing the tagged target nucleotide sequences of each of the        S samples to produce a mixture of tagged target nucleotide        sequences;    -   c) splitting the mixture into a plurality of reaction sites;    -   d) adding different primer pairs to different reaction sites,        wherein each different primer pair includes a primer that        hybridizes to a different sample tag;    -   e) amplifying the tagged target nucleotide sequence from a        different sample in each reaction site; and/or    -   f) detecting the presence of the tagged target nucleotide;        -   wherein step a) includes reaction with a tagged primer that            includes the sample-specific tag but does not include the            target nucleotide sequence.

The tagged primer may be at a higher concentration (e.g., at least 5times higher, at least 10 times higher, or at least 20 times higher)than the tagged target-specific primer. The method may include atarget-specific primer that does not include the tag and is the reversecomplement to a portion of the target nucleotide sequence. Step a) mayfurther include reverse transcribing the target nucleotide sequenceusing the target-specific primer. The tagged target-specific primer mayinclude uracil, e.g., when the method further includes adding a uracilDNA-glycosylase (UDG) to the mixture of tagged target nucleotidesequences to cleave (i.e., degrade) the tagged target-specific primer.

A kit for detecting at least one target nucleic acid in a plurality ofsamples, may therefore include:

a tagged target-specific primer for each of S samples, wherein eachtagged target-specific primer includes a sample-specific tag and atarget-specific sequence, and

a tagged primer that includes the sample-specific tag but does notinclude the target nucleotide sequence.

1-240. (canceled)
 241. An integrated microfluidic device comprising: anarray of reaction sites, wherein individual reaction sites of the arrayof reaction sites comprises an assay chamber and a sample chamber, andwherein sample inlets provide sample to the sample chambers and assayinlets provide assay reagents to the assay chambers; and a plurality ofsample processing unit cells comprising a plurality of sample processingsites, wherein the unit cell is in fluidic communication with aplurality of different reagent inlets, wherein the plurality of sampleprocessing sites comprise a plurality of chambers, and whereinindividual unit cells further comprises at least one column configuredto retain beads; wherein sample inlets to the array are downstream ofthe plurality of sample processing sites of the plurality of unit cells.242. The device of claim 241, wherein the plurality of reagent inletsshare a common channel to each unit cell.
 243. The device of claim 241,further comprising a multiplexor configured to control which reagentinlet is used to load a processing site of the unit cell.
 244. Thedevice of claim 241, wherein the plurality of sample processing sitescomprise a plurality of loops.
 245. The device of claim 241, whereineach unit cells comprises a plurality of valves configured to controlthe unit cell.
 246. The device of claim 245, wherein the plurality ofvalves are configured to place sample processing locations in isolationor in communication with one another.
 247. The device of claim 245,wherein the plurality of valves are configured to drive mixing atdifferent locations, wherein the unit cell comprises a peristaltic pump.248. The device of claim 241, wherein the column comprises a sievearchitecture providing a plurality of openings through which fluid mayflow but beads larger than the opening may be retained.
 249. The deviceof claim 241, wherein individual unit cells comprise at least twocolumns.
 250. The device of claim 241, wherein the microfluidic deviceis an elastomeric microfluidic device.
 251. A method of performing anassay on an integrated microfluidic device, comprising: loading beadsinto a column of a unit cell from a shared inlet; capturing samplebiomolecules of interest on the beads; washing the beads; elutingcaptured biomolecules into a first chamber; loading preamp mastermixinto a second chamber; performing a preamplification reaction; loadingamplicon from preamplification reaction into sample chambers; loading anassay mix into assay chambers; and mixing at least a fraction of thecontents of the sample chamber and assay chamber.
 252. The method ofclaim 251, wherein the step of capturing is after the step of loading.253. The method of claim 251, wherein the preamp master mix comprisesreverse transcriptase and a polymerase, and wherein the preamp mastermixcomprises primer pairs to a plurality of different target nucleotidesequences.
 254. The method of claim 253, wherein the presence of eachtarget nucleotide sequence is detected by qPCR after the step of mixing.255. The method of claim 251, wherein the beads comprise an affinityreagent, and wherein the affinity reagent is an antibody.
 256. Themethod of claim 251, wherein the beads are functionalized with singlestranded DNA sequences that specifically hybridize the one or moretarget nucleotide sequences.
 257. The method of claim 256, wherein theone or more target nucleotide sequences comprise a viral RNA sequence.258. The method of claim 257, wherein the viral RNA sequence is aSARS-CoV-2 RNA sequence, wherein the one or more target nucleotidesequences comprise at least two of N1, N2, N3 and RP (Rnase P)SARS-CoV-2 sequences, and further comprising detecting the at least twoof N1, N2, N3 and RP (Rnase P) SARS-CoV-2 sequences in separate reactionsites.
 259. The method of claim 251, wherein a unit cell comprisesmultiple columns each loaded with beads that capture a differentbiomolecule of interest.
 260. The method of claim 251, wherein the beadscomprise an antibody that binds to a viral particle, the method furthercomprising detecting the presence of the viral particle by immuno-PCR.261. The method of claim 251, wherein a plurality of different proteinsare detected for each of a plurality of different samples, whereindetecting is in the unit cell or wherein detecting is in an array ofreaction sites downstream of the unit cell, and wherein detecting is byqPCR.